Fluorescent polymeric materials containing lipid soluble rhodamine dyes

ABSTRACT

Fluorescent polymeric materials are disclosed comprising a polymer and one or more lipid soluble rhodamine dyes. The materials are especially useful in the preparation of multicolored microparticles, especially multicolored polystyrene microparticle, for use in the multiplexed analysis of a plurality of analytes in a single sample. When excited by a light source, the materials give off a unique emission based on the nature, concentration and ratio of the dyes therein. Methods of preparing and using said materials are also disclosed.

1. CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/837,983, filed May 4, 2004, which claims benefit under 35 U.S.C. §119(e) to U.S. provisional patent application No. 60/469,030, filed May9, 2003, entitled “Fluorescent Polymeric Materials Containing LipidSoluble Rhodamine Dyes,” the disclosures of which are incorporatedherein by reference.

2. BACKGROUND OF THE INVENTION

1. Field Of The Invention

Fluorescent polymeric particles that comprise a polymer and one or morelipid soluble rhodamine dyes are described herein.

2. Description of Related Art

Dyed polymeric materials are known in the art. However, there remains anever present need to develop improved and more diverse dyed polymericmaterials for use in, among other things, the multiplexed analysis of aplurality of analytes in a single assay. Specifically, there is a needfor fluorescent polymeric materials that contain dyes that exhibitenhanced fluorescence, enhanced retention characteristics, and highphotostability without significantly sacrificing other desirableproperties. These and other needs are met by the materials describedherein.

3. SUMMARY OF THE INVENTION

Fluorescent polymeric materials that comprise a polymer and one or morelipid-soluble rhodamine dyes are described herein. Optionally, one ormore additional dyes may be present. Optionally, one or more additionalmaterials may be present including biomolecules as well as compoundsthat aid in material separation such as paramagnetic compounds.

The polymer component may be any polymer that swells in an organicsolvent or solvent system that is used to dissolve a lipid solublerhodamine dye. Illustrative organic solvents include ethyl acetate anddichloromethane. In one embodiment, the polymer component is alsosufficiently hydrophobic that it shrinks in alcoholic solvent, e.g.,isopropanol.

Non-limiting examples of useful polymers include polymers polymerizedfrom one or more substituted or unsubstituted versions of the followingmonomers: styrene; acrylate; alkyl acrylate; methacrylate; alkylmethacrylate; acrylonitrile; alkyl acrylonitrile; esters; acetates;amides; alcohols; isocyanates; acrolein; dimethylsiloxane; butadiene;isoprene; urethane; vinylacetate; vinylchloride; vinylpyridine;vinylbenzylchloride; vinyltoluene; vinylidene chloride; and mixturesthereof. The polymer may be crosslinked or uncrosslinked, and may takeon virtually any shape or form, ranging from amorphous forms tostructured forms like sheets, discs, pellets, beads, etc. In oneembodiment, the polymer is a crosslinked polystyrene bead or particle.

The polymer component may be “activated” to include functional groups,such as carboxylate and/or amine groups, suitable for attaching thefluorescent polymeric material to another substance, such as, forexample, biomolecules, haptens, drugs, poisons, vitamins, antigens andpathogens. Alternatively, the polymer component may include precursorsof such functional groups that can be modified or transformed into suchgroups via chemical or other means. Activation of the polymer componentto include such groups may occur before, during or after incorporationof the lipid soluble rhodamine dyes and/or other optional dyes.

The polymer component can include substances useful in the manipulationof the fluorescent polymeric material. For example, paramagneticcompounds may be present in, or on, the polymer that allow the polymerto be collected by a magnet. These compounds can be included during orafter polymer formation. These substances can be physically entrappedin, or coated onto, the polymer component. In one embodiment, one ormore paramagnetic compounds are physically entrapped within the polymercomponent during polymerization

The polymer component may comprise one or more additional substances.For example, biomolecules, haptens, drugs, poisons, vitamins, antigensand pathogens can be absorbed, adsorbed or covalently attached to thepolymer component which can, for example, enhance the polymercomponent's ability to capture an analyte. In one embodiment, one ormore biomolecules are covalently attached to the polymer component atsome point after the formation of the polymer component.

It will be evident to those skilled in the art that the polymersdiscussed above are readily available from a number of alternativecommercial sources, e.g., Bangs Laboratories, Dynal, Sperotech, andPolyscience. Commercially available products that are available fromthese sources include, among other things, activated polymers,paramagnetic polymers, activated paramagnetic polymers, and polymerscontaining one or more biomolecules. For example, Bangs Laboraties sellsthe following products: “plain (hydrophobic) polystyrene microspheres”of various sizes (catalog codes PS02N, PS03N, PS04N, PS05N, PS06N,PS07N, PS08N, PS00N); “carboxylate-modified microspheres” of varioussizes (catalog codes PC02N, PC03N, PC04N, PC05N, PC06N, PC07N, PC08N andPC00N); “amino-modified microspheres” of various sizes (catalog codesPA02N, PA03N, PA04N, PA05N, PA06N, and PA00N); “classical magneticmicrospheres” having carboxlic or amino functionality (catalog codesMC02N, MC03N, MC04N, MC05N, and MCOON); “encapsulated magneticmicrospheres” with carboxylic and amino surface groups (catalog codesME01N, ME02N, ME03N, and ME00N); and “protein-activated” or“protein-coated” mirospheres (catalog codes CM01N, CM02N, CM03N, CP01N,CP02N and CP03N). Similarly, Dynal sells Dynabeads®, a uniform,superparamagnetic, monodisperse polymer bead that can be uncoated orprecoated with specific ligands. Dynabeads® are available in threedifferent diameters, namely, 1 μm (Dynabeads® MyOne™ Streptavidin), 2.8μm (Dynabeads®(V M-280 and Dynabeads® M-270) and 4.5 μm (Dynabeads®M-450 and Dynabeads® M-500).

The fluorescent polymeric materials additionally comprise one or morelipid-soluble rhodamine dyes. Rhodamines are a well-known class of dyecharacterized by three “main” features: a parent xanthene ring, anexocyclic amine group and an exocyclic imminium group. In mostrhodamines, the exocyclic amine and imminium groups are attached to the3- and 6-carbons of the parent xanthene ring. However, “extendedrhodamines” are known in which the parent xanthene ring includes a 3,4-and/or a 5,6-benzo substituent (see, e.g., U.S. Pat. No. 6,248,884). Insuch “extended rhodamines” the exocyclic amine and imminium groups areattached to any present benzo substituents, as illustrated in U.S. Pat.No. 6,248,884.

As is well known, the parent xanthene ring of the rhodamines may besubstituted at any and all of the aromatic carbons. Non-limitingexamples of substituent groups that may be included in rhodamines atthese positions include hydrogen, amino, hydroxy, alkoxy, mercapto,alkylthio, halo, haloalkyl, cyano, isocyano, cyanato, mercaptocyanato,nitroso, nitro, azido, sulfeno, sulfinyl, sulfino, sulfonyl, sulfonicacid, sulfonic ester, substituted and unsubstituted sulfinamoyl,substituted and unsubstituted sulfamoyl, aldehydes, ketones, carboxylicacid, carboxylic acid ester, amido, substituted or unsubstitutedamidino, alkyl or heteroalkyl, aryl or heteroaryl, and arylalkyl orheteroarylalkyl. In addition, the substituents at the 2-, 4-, 5- and/or7-carbon positions, or the corresponding portion of an extendedrhodamine, may be fused to the nitrogen atom of their respectiveadjacent amine or imminium groups to form rhodamines in which the amineand/or imminium nitrogens are included in ring structures (see, e.g.,U.S. Pat. Nos. 5,231,191, 5,410,053, 5,750,409, 5,847,162, 5,936,087,6,008,379, 6,372,907, and 6,248,884). Rhodamines may also include benzo,naphtho or other polycyclic aryleno substituents fused to the 1,2 and/or7,8 carbons, yielding benzo or naphtho rhodamines (see, e.g., U.S. Pat.Nos. 5,840,999, 5,847,162, 5,936,087, 6,008,379 and 6,248,884). Thecarbons of such benzo, naphtho or polycyclic aryleno substituents may befurther substituted with, for example, one or more of the substituentgroups described above.

Rhodamines may also be substituted at the 9-carbon (C-9 position) of theparent xanthene ring. In one class of rhodamines, the C-9 position issubstituted with a phenyl group which may be further substituted orunsubstituted. Often, such rhodamines include a carboxylate or sulfonategroup on the C-9 phenyl ortho to its point of attachment to theremainder of the ring. The remaining groups on the phenyl ring may befurther substituted with substituent groups such as those previouslydescribed above. Many different types of rhodamines of this class areknown and described (see, e.g., U.S. Pat. Nos. 5,231,191, 5,366,860,5,410,053, 5,750,409, 5,840,999, 5,847,162, 5,936,087, 6,008,379 and6,248,884).

In another class of rhodamines, the 9-carbon is substituted with anon-aromatic substituent, such as for example a hydrogen, alkyl, halo,haloalkyl or nitrile group. Rhodamines of this class are exemplified bythe rhodamines described in U.S. Pat. Nos. 6,008,379 and 6,248,884.

Any of these rhodamines, if made lipid soluble, can be used in thefluorescent polymeric materials described herein. The degree of lipidsolubility necessarily varies as a function of the polymer utilized, theaqueous solvent or solvent system employed in the assay in which thefluorescent polymeric material is used, and the conditions (e.g., time,temperature, pressure, pH, etc.) under which the assay is run. Suitabledegrees of lipid solubility are easily determined by methods known inthe art. For example, suitable lipid solubility can be determined by apartition test wherein a known quantity of dye in organic solvent iscombined with the aqueous solvent or solvent system used in the assay.If a partition results and, under the conditions used in the assay,there is no appreciable crossing by the dye into the solvent or solventsystem, then the dye is sufficiently lipid soluble. Put another way, thelipid soluble rhodamine dye should be sufficiently lipid soluble suchthat it is capable of being imbibed into the polymer when dissolved inan organic solvent or solvent system and, when the dyed polymer issubjected to the aquesous conditions of the assay, the dye should resistleaching out of the polymer to any degree that significantly impacts thefluorescent signature of the dye imbibed polymer or the results of theassay.

Generally, useful lipid-soluble rhodamines are rhodamines that aresubstituted at one or both of the exocyclic amine and/or imminiumnitrogens with a lipophilic substituent group designed to impart theresultant rhodamine dye with lipophilic characteristics or properties.Thus, useful lipid-soluble rhodamines may include one or two lipophilicsubstituents at the exocyclic amine nitrogen and/or one or twolipophilic substituents at the exocyclic imminium nitrogen. Thelipophilic substituents, whether attached to the same or differentnitrogen atoms, may be the same or different. In one embodiment, thelipid-soluble rhodamine is a rhodamine in which the exocyclic amine andexocyclic imminium nitrogens are each substituted with identicallipophilic groups.

Lipid-soluble rhodamines may include lipophilic substitutents at otherpositions, as well. In certain embodiments, it may be desirable to“mask” polar substituents on a particular rhodamine with lipophilicsubstituent groups to alter the lipid-solublity of the resultantrhodamine. For example, rhodamines which include a C-9 phenylsubstituted at the ortho position with a carboxylate or sulfonate groupmay be reacted with a lipophilic alcohol or amine, such as an alkyl,aryl or arylalkyl alcohol or amine, to yield an ortho ester or amidethat contributes to the net lipophilic properties of the resultantrhodamine dye.

Lipophilic substituents are groups that impart the resultant rhodaminedye with lipophilic character or properties as denoted above. The natureof each lipophilic substituent is not critical, as long as the resultantrhodamine dye is lipid soluble. Non-limiting examples of suitablelipophilic substituents include unsubstituted (C4-C20) alkyls, (C5-C40)aryls, and (C6-C40) arylalkyls. Depending on the number of methylene andmethine units in the lipophilic substituent, the lipophilic substituentmay also include pendant or internal hydrophilic groups. For example, alipophilic substitutent may include one or more internal heteroatoms,such as one or more internal O, S, N or NH groups. As another example, alipophilic substituent may include one or more pendant polar orhydrophilic substituents, such as one or more pendant halogen, —OH, —SH,—NH₂, —C(O)OH, —C(O)NH₂ or other polar or hydrophilic groups. Thus,lipophilic substituents may also include substituted (C4-C20) alkyl,substituted (C5-C40) aryls and substituted (C6-C40) arylalkyls, as wellas substituted and unsubstituted (C4-C20) heteroalkyl, substituted andunsubstituted (C5-C40) heteroaryls and substituted and unsubstituted(C6-C40) arylalkyls. As a specific example, if the rhodamine contains aC-9 phenyl ring, (C10-20) alkyl esters and alkyl amides are oftenemployed in one or both of the positions ortho to the point of thephenyl ring's attachment. The number of internal or pendant polar orhydrophilic groups that may be included in a lipophilic substituent willdepend upon, among other factors, the number of methylene or methinegroups included in the lipophilic substitutent and the number oflipophilic substituents on the rhodamine dye. The nature and number oflipophilic groups necessary to make a rhodamine lipid soluble can varyfrom molecule to molecule, and will be apparent to those of skill in theart.

In one embodiment, the lipid-soluble rhodamine dyes useful in thefluorescent polymeric materials include any lipid-soluble dye comprisingone of the following “core” structures:

where R¹ R², R^(2′), R^(3′), R^(3″), R⁴, R^(4′), R^(5′), R⁵, R^(6″),R^(6′), R^(7′), R⁷, R⁸, R⁹, R¹¹ R¹², R¹³, R¹⁴ and R¹⁵ are selected fromhydrogen or the same or different substituent groups, at least one ofR^(3′), R^(3″), R^(6″) and R^(6′) is a lipophilic substituent, and R⁹ instructure (II) is a nonaromatic substituent. In one embodiment, all ofthe substituents, whether they represent R¹, R², R^(2′), R³, R^(3″), R⁴,R^(4′), R^(5′), R⁵, R^(6″), R^(6′), R^(7′), R⁷, R⁸, R⁹, R¹¹, R¹², R¹³,R¹⁴ or R¹⁵, contain no more than 40 atoms. In another embodiment, thesubstituent groups at one or more of R², R^(2′), R⁴, R^(4′) R^(5′), R⁵,R^(7′), R⁷, R⁹, R¹¹, R¹², R¹³, R¹⁴, and R¹⁵ are selected to “tune” thespectral features of the lipid-soluble rhodamine dye. Usually, forsteric reasons, R¹ and R⁸ in structures (I), (III) and (IV) are notsimultaneously pendant or fused benzo, naphtho or polycyclic arylenorings.

In embodiments in which the lipid-soluble rhodamine dye is attached tothe polymer via a covalent linkage, one or more of R¹, R², R^(2′), R⁴,R⁵, R⁷, R⁸, R¹¹, R¹², R¹³, R¹⁴ and R¹⁵ may comprise a linking groupsuitable for effecting such covalent attachment. The linking group(s)may be attached directly to the remainder of the molecule, or may bespaced away from the molecule via a linker or spacer, as is well knownin the art.

It has been discovered that certain types of C-9 phenyl substitutedlipid soluble rhodamines have especially good fluorescent properties.These lipid soluble rhodamines depart from known C-9 phenyl substitutedrhodamines in many ways, including the fact that they do not possess anortho carboxylic acid or an ortho sulfonic acid moiety, or an amide,acid halide or salt derivative thereof. Often these lipid solublerhodamines have symmetrical substitutions on the C-9 phenyl ring. In oneembodiment, these lipid soluble rhodamine dyes comprise one of corestructures (I), (III), and (IV) above where at least one of R¹¹ and R¹⁵are selected, independently of one another, from (C1-C20) alkyl orheteroalkyl, (C1-C20) alkoxy, halo, (C1-C20) haloalkyl, amino, mercapto,(C1-C20) alkylthio, cyano, isocyano, cyanato, mercaptocyanato, nitroso,nitro, azido, sulfeno, sulfinyl, and sulfino, and the remainder of R¹²,R¹³ and R¹⁴ are selected, independently of one another, from hydrogen,(C1-C20) alkyl or heteroalkyl, (C1-C20) alkoxy, halo, (C1-C20)haloalkyl, amino, mercapto, (C1-C20) alkylthio, cyano, isocyano,cyanato, mercaptocyanato, nitroso, nitro, azido, sulfeno, sulfinyl, andsulfino. Alternatively, R¹¹ and R¹⁵ are each the same substituentselected from (C1-C20) alkyl or heteroalkyl, (C1-C20) alkoxy, halo,(C1-C20) haloalkyl, amino, mercapto, (C1-C20) alkylthio, cyano,isocyano, cyanato, mercaptocyanato, nitroso, nitro, azido, sulfeno,sulfinyl, and sulfino, and the remainder of R¹², R¹³ and R¹⁴ areselected, independently of one another, from hydrogen, (C1-C20) alkyl orheteroalkyl, (C1-C20) alkoxy, halo, (C1-C20) haloalkyl, amino, mercapto,(C1-C20) alkylthio, cyano, isocyano, cyanato, mercaptocyanato, nitroso,nitro, azido, sulfeno, sulfinyl, and sulfino. In yet another embodiment,R¹¹ and R¹⁵ are each the same substituent as just described and R¹², R¹³and R¹⁴ are either unsubstituted or substituted in the same manner asR¹¹ and R¹⁵. In still another embodiment, R¹¹ and R¹⁵ are each the samehalo group, such as chlorine or fluorine, and R¹², R¹³ and R¹⁴ are eachhydrogen or the same halo group selected for R¹¹ and R¹⁵.

The lipid soluble rhodamine dyes may be part of an energy transfer(“ET”) network comprising, for example, two to four dyes covalentlyattached to one another that transfer energy to generate a longerStoke's shift. One example of an ET network would be a fluorescenceresonance energy transfer (“FRET”) dye. In other words, the lipidsoluble rhodamine dyes may be part of series of dyes that are covalentlyattached to one another wherein at least one of the dyes is a lipidsoluble rhodamine. Linkages for covalently attaching rhodamine dyes toother dyes are known in the art, as are suitable locations forattachment to the rhodamine dyes (see, e.g., U.S. Pat. Nos. 5,800,996and 5,863,727). In one embodiment, each dye in the energy transfernetwork is within 5 to 100 Å of the neighboring dye or dyes in thenetwork to which it is covalently attached. In such embodiments, thelipid soluble rhodamine dye can be the donor, acceptor, or anintermediate dye in the network.

The lipid soluble rhodamine dyes may be used alone or in combinationwith one another. In addition, the lipid soluble rhodamine dyes may beused in combination with any other class of dye capable of fluorescingin the polymer employed. In one embodiment where the dyes are used incombination, the dyes are selected to impart the polymer material with aunique spectral signature or “bar code.”

The aforementioned dyes are either imbibed into, or covalently bound to,the polymer. In one embodiment, the dyes are imbibed into the polymerwithout covalent attachment by introducing the dyes to the polymer in anorganic solvent.

The fluorescent polymeric materials are useful in a wide variety ofapplications. For example, the fluorescent polymeric materials may beused as reference standards for fluorescence-based instruments, asbiological tracers and in the detection and/or analysis of biologicalmolecules. Numerous biological assays are known that employ biologicalor other molecules attached to solid supports. The fluorescent polymericmaterials may be used as the solid support in any of these assays.

As a specific example, the fluorescent polymeric materials areespecially beneficial in the formation of multicolored particles for usein multiplexed analysis of a plurality of analytes in a single assay.For example, a first polymeric particle or population of polymericparticles may be dyed with one or more fluorescent dyes, at least one ofwhich is a lipid-soluble rhodamine dye as described above, at specifiedconcentrations and ratios to create a first particle or particlepopulation having a unique, discernable, fluorescence signature or “barcode.” The first particle or particle population can then be modified tohave a specific reactivity with one or more analytes. For example, theparticle or particle population can be covalently attached to a moleculeof interest, such as a potential drug candidate or a biological moleculesuch as an amino acid, a peptide, a protein, a nucleoside or nucleotide,an oligonucleotide, a polynucleotide, a carbohydrate or other moleculeof interest. As specific examples, the first particle or particlepopulation can be attached to an oligonucleotide which specificallyhybridizes to a polynucleotide sequence of interest or, alternatively,to a peptide known or thought to inhibit an enzyme of interest. Thus,following modification, the spectral “bar code” of the first particle orfirst particle population corresponds to or correlates with the specificreactivity of the particle or particle population. In connection withthe specific examples, the spectral “bar code” of the first particle orparticle population corresponds to or correlates with the identity ofthe molecule of interest bound thereto.

The first particle or particle population may then be mixed with one ormore additional particles or particle populations, each of which has adifferent, spectrally resolvable or discernable “bar code” due todifferences in the identities, concentrations and/or ratios of thefluorescent dyes therein, and which correlate with different knowreactivities. This forms a multicolored particle mixture in which thespectral “bar code” of a particular particle identifies the reactivityof the particle.

Depending upon the particular application, the particle mixture may becontacted with one or more analytes under conditions in which theanalytes can interact with the particles. The analytes are typicallymolecules that are potentially capable of specifically binding themolecules attached to the particles. The analyte may be labeled with afluorescent reporter dye that is spectrally resolvable from the “barcodes” of the particles. For example, if the bar code dyes emit in thered region of the spectrum, the analyte may be labeled with afluorescent dye that emits in the green region of the spectrum.Alternatively, the analyte may be unlabeled, or modified to include arecognition moiety recognized by another molecule (e.g., biotin or anantibody epitope) and the fluorescent reporter dye attached to asecondary molecule that specifically recognizes the analyte, the boundanalyte or the recognition moiety. Following contact and washing toremove unbound analyte and/or secondary molecule, fluorescence from thereporter dye identifies the presence of bound analyte on particularparticles. The fluorescent bar codes of these particles identifies theirspecific reactivities. In connection with the specific examples, thefluorescent bar code of a particular particle identifies the moleculebound thereto.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a first illustrative synthesis for the formation of one typeof lipid soluble rhodamine dye.

FIG. 2 is a second illustrative synthesis for the formation of a secondtype of lipid soluble rhodamine dye.

FIG. 3 is a third illustrative synthesis for the formation of a thirdtype of lipid soluble rhodamine dye.

FIG. 4 provides a graph of normalized intensity versus wavelength forexemplary fluorescent dye imbibed beads.

5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 5.1 NumberingSystem

For the purposes of the present application, carbon atoms in the parentrhodamine rings, or extended versions thereof, are numbered in themanner illustrated below:

5.2 Definitions

As used herein, the following terms are intended to have the followingmeanings:

“Fluorescent Dye” or “Fluorescer” or “Fluorochrome” or “Fluorophore” asused interchangeably herein refer to molecules that absorbelectromagnetic radiation at one wavelength and emit electromagneticradiation at another wavelength in passing from a higher to a lowerelectronic state.

“Rhodamine” refers to any compound that comprises a parent rhodaminering or an extended rhodamine ring, as set forth above, including anysubstituted versions of the same, wherein substitutions can be made atany one or all of the 1-, 2-, 2′-, 4-, 4′-, 5′-, 5-, 7′-, 7-, 8- and9-carbons and/or at any one or both of the exocyclic amino and iminonitrogens.

“Polymer,” as used herein, refers not only to homopolymers but also tocopolymers, terpolymers, and the like, whether branched or linear andwhether crosslinked or uncrosslinked.

“Poly-,” when used as a prefix in the name of a polymer, refers to apolymer made up in the majority of the monomer or monomers that followthe prefix.

“Particles” as used herein refers to solid masses that are spherical orirregular in shape.

“Particle size” or “Particle Diameter” as used interchangeably hereinrefers to mean particle diameter. As known in the art, there are manymethods for measuring particle size including sieving, centrifugation,light scattering, image analysis, electrozone sensing, and x-rayscattering. Each method can produce different results. Accordingly,whenever a particle size range is specified in this description itrefers to a particle size as measured by light scattering.

“Microparticles” as used herein refer to particles having a particlesize anywhere between 0.01 to 1000 micrometers as measured by lightscattering.

“Biomolecule” as used herein refers to a molecule of a type typicallyfound in a biological system, whether such molecule is naturallyoccurring or the result of some external disturbance of the system(e.g., a disease, poisoning, genetic manipulation, etc.), as well assynthetic analogs and derivatives thereof. Non-limiting examples ofbiomolecules include amino acids (naturally occurring or synthetic),peptides, polypeptides, glycosylated and unglycosylated proteins (e.g.,polyclonal and monoclonal antibodies, receptors, interferons, enzymes,etc.), nucleosides, nucleotides, oligonucleotides (e.g., DNA, RNA, PNAoligos), polynucleotides (e.g., DNA, cDNA, RNA, etc.), carbohydrates,hormones, haptens, steroids, toxins, etc. Biomolecules may be isolatedfrom natural sources, or they may be synthetic. “Analyte” is a moleculeor substance to be measured or assayed. Depending on the nature of theassay, an analyte can be either a molecule or a substance attached tothe polymer or a molecule or substance in solution.

“Alkyl” by itself or as part of another substituent refers to asaturated or unsaturated branched, straight-chain or cyclic monovalenthydrocarbon radical having the stated number of carbon atoms (i.e.,C1-C6 means one to six carbon atoms) that is derived by the removal ofone hydrogen atom from a single carbon atom of a parent alkane, alkeneor alkyne. Typical alkyl groups include, but are not limited to, methyl;ethyls such as ethanyl, ethenyl, ethynyl; propyls such as propan-1-yl,propan-2-yl, cyclopropan-1-yl, prop-1-en-1-yl, prop-1-en-2-yl,prop-2-en-1-yl, cycloprop-1-en-1-yl; cycloprop-2-en-1-yl,prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butyls such as butan-1-yl,butan-2-yl, 2-methyl-propan-1-yl, 2-methyl-propan-2-yl, cyclobutan-1-yl,but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl,but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl,cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl,but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like. Wherespecific levels of saturation are intended, the nomenclature “alkanyl,”“alkenyl” and/or “alkynyl” is used, as defined below. In preferredembodiments, the alkyl groups are (C1-C20) alkyl.

“Heteroalkyl,” by itself or as part of another substituent refers to analkyl in which one or more of the carbon atoms are each independentlyreplaced with the same or different heteratoms or heteroatomic groups.Typical heteroatoms and/or heteroatomic groups which can replace thecarbon atoms include, but are not limited to, —O—, —S—, —S—O—, —NR^(m)—,—P(O)O—, —S(O)—, —S(O)₂—, —S(O)NR^(m)—, —S(O)₂NR^(m)—, and the like,including combinations thereof, where each R^(m) is independentlyhydrogen or (C1-C6) alkyl.

“Aryl” by itself or as part of another substituent refers to amonovalent aromatic hydrocarbon group having the stated number of carbonatoms (i.e., C5-C15 means from 5 to 15 carbon atoms) derived by theremoval of one hydrogen atom from a single carbon atom of a parentaromatic ring system. Typical aryl groups include, but are not limitedto, groups derived from aceanthrylene, acenaphthylene,acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene,fluoranthene, fluorene, hexacene, hexaphene, hexylene, as-indacene,s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene,ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene,phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene,rubicene, triphenylene, trinaphthalene, and the like, as well as thevarious hydro isomers thereof. In preferred embodiments, the aryl groupis (C5-C15) aryl, with (C5-C10) being even more preferred. Particularlypreferred aryls are phenyl and naphthyl.

“Arylaryl” by itself or as part of another substituent refers to amonovalent hydrocarbon group derived by the removal of one hydrogen atomfrom a single carbon atom of a ring system in which two or moreidentical or non-identical parent aromatic ring systems are joineddirectly together by a single bond, where the number of such direct ringjunctions is one less than the number of parent aromatic ring systemsinvolved. Typical arylaryl groups include, but are not limited to,biphenyl, triphenyl, phenyl-naphthyl, binaphthyl, biphenyl-naphthyl, andthe like. Where the number of carbon atoms in an arylaryl group arespecified, the numbers refer to the carbon atoms comprising each parentaromatic ring. For example, (C5-C15) arylaryl is an arylaryl group inwhich each aromatic ring comprises from 5 to 15 carbons, e.g., biphenyl,triphenyl, binaphthyl, phenylnaphthyl, etc. Preferably, each parentaromatic ring system of an arylaryl group is independently a (C5-C15)aromatic, more preferably a (C5-C10) aromatic. Also preferred arearylaryl groups in which all of the parent aromatic ring systems areidentical, e.g., biphenyl, triphenyl, binaphthyl, trinaphthyl, etc.

“Arylalkyl” by itself or as part of another substituent refers to anacyclic alkyl group in which one of the hydrogen atoms bonded to acarbon atom, typically a terminal or sp³ carbon atom, is replaced withan aryl group. Typical arylalkyl groups include, but are not limited to,benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl,2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl,2-naphthophenylethan-1-yl and the like. Where specific alkyl moietiesare intended, the nomenclature arylalkanyl, arylakenyl and/orarylalkynyl is used. In preferred embodiments, the arylalkyl group is(C6-C21) arylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of thearylalkyl group is (C1-C6) and the aryl moiety is (C5-C15). Inparticularly preferred embodiments the arylalkyl group is (C6-C13),e.g., the alkanyl, alkenyl or alkynyl moiety of the arylalkyl group is(C1-C3) and the aryl moiety is (C5-C10).

“Heteroaryl” by itself or as part of another substituent refers to amonovalent heteroaromatic group having the stated number of ring atoms(e.g., “5-14 membered” means from 5 to 14 ring atoms) derived by theremoval of one hydrogen atom from a single atom of a parentheteroaromatic ring system. Typical heteroaryl groups include, but arenot limited to, groups derived from acridine, benzimidazole,benzisoxazole, benzodioxan, benzodiaxole, benzofuran, benzopyrone,benzothiadiazole, benzothiazole, benzotriazole, benzoxazine,benzoxazole, benzoxazoline, carbazole, β-carboline, chromane, chromene,cinnoline, furan, imidazole, indazole, indole, indoline, indolizine,isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline,isothiazble, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine,phenanthridine, phenanthroline, phenazine, phthalazine, pteridine,purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine,pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and thelike, as well as the various hydro isomers thereof. In preferredembodiments, the heteroaryl group is a 5-14 membered heteroaryl, with5-10 membered heteroaryl being particularly preferred.

“Heteroaryl-Heteroaryl” by itself or as part of another substituentrefers to a monovalent heteroaromatic group derived by the removal ofone hydrogen atom from a single atom of a ring system in which two ormore identical or non-identical parent heteroaromatic ring systems arejoined directly together by a single bond, where the number of suchdirect ring junctions is one less than the number of parentheteroaromatic ring systems involved. Typical heteroaryl-heteroarylgroups include, but are not limited to, bipyridyl, tripyridyl,pyridylpurinyl, bipurinyl, etc. Where the number of atoms are specified,the numbers refer to the number of atoms comprising each parentheteroaromatic ring systems. For example, 5-15 memberedheteroaryl-heteroaryl is a heteroaryl-heteroaryl group in which eachparent heteroaromatic ring system comprises from 5 to 15 atoms, e.g.,bipyridyl, tripuridyl, etc. Preferably, each parent heteroaromatic ringsystem is independently a 5-15 membered heteroaromatic, more preferablya 5-10 membered heteroaromatic. Also preferred are heteroaryl-heteroarylgroups in which all of the parent heteroaromatic ring systems areidentical.

“Heteroarylalkyl” by itself or as part of another substituent refers toan acyclic alkyl group in which one of the hydrogen atoms bonded to acarbon atom, typically a terminal or sp³ carbon atom, is replaced with aheteroaryl group. Where specific alkyl moieties are intended, thenomenclature heteroarylalkanyl, heteroarylakenyl and/orheteroarylalkynyl is used. In preferred embodiments, the heteroarylalkylgroup is a 6-21 membered heteroarylalkyl, e.g., the alkanyl, alkenyl oralkynyl moiety of the heteroarylalkyl is (C1-C6) alkyl and theheteroaryl moiety is a 5-15-membered heteroaryl. In particularlypreferred embodiments, the heteroarylalkyl is a 6-13 memberedheteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety is (C1-C3)alkyl and the heteroaryl moiety is a 5-10 membered heteroaryl.

“Halogen” or “Halo” by themselves or as part of another substituent,unless otherwise stated, refer to fluoro, chloro, bromo and iodo.

“Haloalkyl” by itself or as part of another substituent refers to analkyl group in which one or more of the hydrogen atoms is replaced witha halogen. Thus, the term “haloalkyl” is meant to includemonohaloalkyls, dihaloalkyls, trihaloalkyls, etc. up to perhaloalkyls.For example, the expression “(C1-C2) haloalkyl” includes 1-fluoromethyl,difluoromethyl, trifluoromethyl, 1-fluoroethyl, 1,1-difluoroethyl,1,2-difluoroethyl, 1,1,1-trifluoroethyl, perfluoroethyl, etc.

The above-defined groups may include prefixes and/or suffixes that arecommonly used in the art to create additional well-recognizedsubstituent groups. As examples, “alkyloxy” or “alkoxy” refers to agroup of the formula —OR^(n), “alkylamine” refers to a group of theformula —NHR^(n) and “dialkylamine” refers to a group of the formula—NR^(n)R^(n), where each R^(n) is independently an alkyl. As anotherexample, “haloalkoxy” or “haloalkyloxy” refers to a group of the formula—ORP, where R^(P) is a haloalkyl.

5.3 The Components Generally

The present invention provides new types of fluorescent polymericmaterials. The fluorescent polymeric materials comprise a polymer thatis either imbibed with, or covalently attached to, one or more lipidsoluble rhodamine dyes and, optionally, one or more additional dyes. Thepolymer may be activated to include functional groups suitable forattaching another substance such as a biomolecule. Alternatively, thepolymer component may include precursors of such functional groups thatcan be modified or transformed into such groups via chemical or othermeans.

The fluorescent polymeric materials can also include one or more othersubstances, such as biomolecules, haptens, drugs, poisons, vitamins,antigens and pathogens, that are absorbed, adsorbed or covalentlyattached to the polymer. These substances can be added, for example, toenhance the polymer component's ability to capture an analyte.Additional substances that may be part of the fluorescent polymericmaterials include compounds that assist in the manipulation of thefluorescent polymeric material. For example, paramagnetic compounds canbe included that permit the fluorescent polymeric material to becollected by a magnet. All of these substances can be physicallyentrapped within, imbibed into, covalently attached to, or coated ontothe polymer component.

5.4 The Polymer Component

The polymer component may be any polymer that can be dissolved in, ormade to swell in, an organic solvent or solvent system that is used todissolve a lipid soluble rhodamine dye. Illustrative organic solventsinclude ethyl acetate and/or dichloromethane. In one embodiment, thepolymer component is sufficiently hydrophobic that it also shrinks inalcoholic solvent, e.g., isopropanol. This assists, for example, inentrapping previously imbibed lipid soluble rhodamine dyes.

Non-limiting examples of polymer components useful in the fluorescentpolymeric materials of the instant invention are polymers polymerizedfrom substituted or unsubstituted versions of the following monomers:styrene; acrylate; alkyl acrylate; methacrylate; alkyl methacrylate;acrylonitrile; alkyl acrylonitrile; esters; acetates; amides; alcohols;acrolein; dimethylsiloxane, butadiene, isoprene, urethane, vinylacetate,vinylchloride, vinylpyridine, vinylbenzylchloride; vinyltoluene;vinylidene chloride; and mixtures thereof. Specific non-limitingexamples of suitable polymers include polystyrene, brominatedpolystyrene, poly(methyl methacrylate), poly(acrylonitrile),polyacrolein, poly(dimethyl siloxane), polybutadiene, polyisoprene,polyurethane, poly(vinyl acetate), poly(vinyl chloride), poly(vinylpyridine), poly(vinyl benzylchloride), poly(vinyl toluene),poly(vinylidene chloride) and poly(divinyl benzene). Other suitablepolymers are known in the art and disclosed, for instance, in U.S. Pat.No. 6,268,222, which is incorporated herein by reference. In oneembodiment of the invention, the polymer is a polystyrene homopolymer ora copolymer of styrene polymerized with one or more other monomers, suchas acrylic acid.

The polymer can be crosslinked or uncrosslinked. The identity and amountof crosslinking agents that can be employed varies based on theselection of the particular polymer in a manner that is readily evidentto those skilled in the art. Crosslinking agents are generally used inamounts ranging from about 0.01 to about 50 percent by weight of thepolymer. In one embodiment, crosslinking agents are employed in amountsranging from 0.05 to 20 percent by weight of the polymer. In anotherembodiment, crosslinking agents are employed in amounts ranging from 1to 5 percent by weight of the polymer. The more crosslinking agentutilized, the less the polymer is able to swell. Illustrativenon-limiting examples of suitable crosslinking agents include divinylbenzene, ethylene glycol dimethacrylate, trimethylol propanetrimethacrylate, and N,N′-methylene-bis-acrylamide, and the like.Basically, any polyfunctional reactive agent can be utilized as acrosslinking agent. In one embodiment, the polymer is a hydrophobiccopolymer of styrene, acrylic acid, and a polyvinyl crosslinking agent.Non-limiting examples of such polymers include those polymerized from85% or more styrene, 0.01 to 15% acrylic acid, and 0.01 to 5% polyvinylcrosslinking agent. Illustrative copolymers are styrene-acrylicacid-divinyl benzene (89:10:1 molar ratio).

The polymer may take on virtually any shape or form, ranging fromamorphous forms to structured forms like sheets, discs, pellets, beads,etc. In one embodiment, the polymer forms particles, and ideallymicroparticles, which substantially retain their shape in aqueoussolvent so that they are useful in biological assays. By microparticlesit is meant that the particles have a mean diameter ranging from about0.01 to about 1000 micrometers. Although the microparticles can have anyparticle size within this range, in one embodiment the particles rangein size from about 0.1 to about 500 micrometers. Alternatively, theparticles may range in size from about 1 to about 200 micrometers. Inanother embodiment the particles range in size from about 1 to about 12micrometers. In yet another embodiment, the particle size is from about1 to about 5 micrometers. The polymer particles may be uniform or ofvariable size and shape. Preferably, the polymer particles are roughlyspherical and of a generally uniform diameter.

Optionally, the polymer component can be “activated” to include areactive functionality (e.g., NHS-ester) capable of forming a chemicalattachment to another substance. Alternatively, the polymer componentcan be “activated” to include groups that have a functionality (e.g.,carboxylic acid) that are known precursors to reactive functionalitiescapable of forming a chemical attachment to another substance.Non-limiting examples of other substances for attachment includebiomolecules, drugs, poisons, vitamins, antigens and pathogens.Activation can be accomplished either by copolymerizing or graftingfunctional monomers into the polymer during polymer formation or bychemically modifying the polymer after formation. The reactive groupsmay be nucleophilic and/or electrophilic functional groups. Non-limitingexamples of suitable nucleophilic reactive groups include any one or acombination of the following: amines/anilines; alcohols/phenols; thiols;hydrazines; and hydroxylamines. Non-limiting examples of suitableelectrophilic reactive groups include any one or a combination of thefollowing: pentafluorophenyl ester; NHS-ester; acrylamides; acyl azides;acyl halides; acyl nitriles; aldehydes or ketones; alkyl halides; alkylsulfonates; anhydrides; aryl halides; aziridines; boronates; carboxylicacids; carbodiimides; diazoalkenes; epoxides; haloacetamides;halotriazines; imido esters; isocyanates; isothiocyanates; maleimides;phorphoramidites; silyl halides; sulfonate esters; and sulfonyl halides.

The identity of any particular activating group will depend upon theparticular application, for example upon the identity of the moleculesto be attached thereto, and will be apparent to those of skill in theart. For example, polymers activated with carboxyl groups are especiallysuitable for attachment to the primary amine groups of proteins oramine-modified oligonucleotides. Such carboxyl groups can be introducedinto the polymer chain through reactive comonomers such as (meth)acrylicacid. In addition, polymers containing groups such as diamines,dihydrazides, mercaptoalkylamines and dimercaptans are useful forattaching various drugs or enzymes.

The nature of the active groups can be selected to manipulate variouscharacteristics of the polymer surface. For example, if desired, activegroups can be selected to make the polymer surface more hydrophilic.

As will be further detailed, the polymer can include paramagneticcompounds such as magnetite (Fe₃O₄). These compounds can be coated ontothe polymer surface during or after formation and/or entrapped into thepolymer during polymerization. For example, one method of incorporatingparamagnetic compounds into the polymer is to form a core of a coreshell polymer, coat the core with a paramagnetic compound, and then formthe shell of the core shell polymer over the paramagnetic compound,thereby entrapping the paramagnetic compound within the finishedpolymer.

As will also be further detailed, the polymer component can comprise oneor more additional substances. For example, biomolecules, haptens,drugs, poisons, vitamins, antigens and pathogens can be absorbed,adsorbed or covalently attached to the polymer component which can, forexample, enhance the polymer component's ability to capture an analyte.In one embodiment, one or more of biomolecules are covalently attachedto the polymer component at some point after the formation of thepolymer component.

It will be evident to those skilled in the art that the polymersdiscussed above are readily available from a number of alternativecommercial sources, e.g., Bangs Laboratories, Dynal, Sperotech, andPolyscience. Commercially available products that are available fromthese sources include, among others, activated polymers, paramagneticpolymers, activated paramagnetic polymers, and polymers containing oneor more biomolecules. For example, Bangs Laboraties sells the followingproducts: “plain (hydrophobic) polystyrene microspheres” of varioussizes (catalog codes PS02N, PS03N, PS04N, PS05N, PS06N, PS07N, PS08N,PS00N); “carboxylate-modified microspheres” of various sizes (catalogcodes PC02N, PC03N, PC04N, PC05N, PC06N, PC07N, PC08N and PC00N);“amino-modified microspheres” of various sizes (catalog codes PA02N,PA03N, PA04N, PA05N, PA06N, and PA00N); “classical magneticmicrospheres” having carboxlic or amino functionality (catalog codesMC02N, MC03N, MC04N, MC05N, and MCOON); “encapsulated magneticmicrospheres” with carboxylic and amino surface groups (catalog codesME01N, ME02N, ME03N, and ME00N); and “protein-activated” or“protein-coated” mirospheres (catalog codes CM01N, CM02N, CM03N, CP01N,CP02N and CP03N). Similarly, Dynal sells Dynabeads®, a uniform,superparamagnetic, monodisperse polymer bead that can be uncoated orprecoated with specific ligands. Dynabeads®(V are available in threedifferent diameters, namely, 1 μm (Dynabeads® MyOne™ Streptavidin), 2.8μm (Dynabeads® M-280 and Dynabeads® M-270) and 4.5 μm (Dynabeads® M-450and Dynabeads®t M-500).

5.5 Lipid Soluble Rhodamine Dyes Generally

The polymer component is imbibed or swelled with, or dissolved in, asolution of one or more lipid soluble rhodamine dyes and, optionally,one or more additional dyes. Alternatively, the lipid soluble rhodaminedyes can be covalently attached to the polymer.

Rhodamines are a well-known class of dye characterized by three “main”features: a parent xanthene ring, an exocyclic amine group and anexocyclic imminium group. In most rhodamines, the exocyclic amine andimminium groups are attached to the 3- and 6-carbons of the parentxanthene ring. However, “extended rhodamines” are known in which theparent xanthene ring includes a 3,4- and/or a 5,6-benzo substituent. Inthis regard, U.S. Pat. No. 6,248,884 is hereby incorporated byreference. In such “extended rhodamines” the exocyclic amine andimminium groups are attached to any present 3,4- and/or 5,6-benzosubstituents.

The parent xanthene ring of the rhodamines may be substituted at the 1-,2-, 2′-, 4-, 4′-, 5-, 5′-, 7-, 7′- and 8-carbon positions. Non-limitingexamples of substituent groups that may be included in rhodamines atthese positions include hydrogen, amino, hydroxy, alkoxy, mercapto,alkylthio, halo, haloalkyl, cyano, isocyano, cyanato, mercaptocyanato,nitroso, nitro, azido, sulfeno, sulfinyl, sulfino, sulfonyl, sulfonicacid, sulfonic ester, substituted and unsubstituted sulfinamoyl,substituted and unsubstituted sulfamoyl, aldehydes, ketones, carboxylicacid, carboxylic acid ester, amido, substituted or unsubstitutedamidino, alkyl or heteroalkyl, aryl or heteroaryl, and arylalkyl orheteroarylalkyl.

In addition, the substituents at the 2- or 2′-, 4-, 5- and/or 7- or7′-carbon positions may be fused to the nitrogen atom of theirrespective adjacent amine or imminium groups to form rhodamines in whichthe amine and/or imminium nitrogens are included in ring structures. Inthis regard, U.S. Pat. Nos. 5,231,191, 5,410,053, 5,750,409, 5,847,162,5,936,087, 6,008,379, 6,372,907, and 6,248,884 are hereby incorporatedby reference. Rhodamines may also include benzo, naphtho or otherpolycyclic aryleno substituents fused to the 1,2 and/or 7,8 carbons,yielding benzo or naphtho rhodamines. In this regard, U.S. Pat. Nos.5,840,999, 5,847,162, 5,936,087, 6,008,379 and 6,248,884 are herebyincorporated by reference. The carbons of such benzo, naphtho orpolycyclic aryleno substituents may be further substituted with, forexample, one or more of the substituent groups described above.

Rhodamines may also be substituted at the 9-carbon (C-9 position) of theparent xanthene ring. In one class of rhodamines, the C-9 position issubstituted with a phenyl group which may be further substituted orunsubstituted. Often, such rhodamines include a carboxylate or sulfonategroup positioned ortho to the point of attachment. The remaining groupson the phenyl ring may be unsubstituted or substituted with substituentgroups such as those previously described above. Rhodamines of thisclass are known and described in U.S. Pat. Nos. 5,231,191, 5,366,860,5,410,053, 5,750,409, 5,840,999, 5,847,162, 5,936,087, 6,008,379 and6,248,884, all of which are hereby incorporated by reference.

In another class of rhodamines, the C-9 position is substituted with anon-aromatic substituent, such as a hydrogen, alkyl, halo, haloalkyl ornitrile group. Rhodamines of this class are described in U.S. Pat. Nos.6,008,379 and 6,248,884, both of which are hereby incorporated byreference.

Any of these rhodamines, if made lipid soluble, can be used in thefluorescent polymeric materials described herein. The degree of lipidsolubility necessarily varies as a function of the polymer utilized, theaqueous solvent or solvent system employed in the assay in which thefluorescent polymeric material will be used, and the conditions (e.g.,time, temperature, pressure, pH, etc.) under which the assay is run.Suitable degrees of lipid solubility are easily determined by methodsknown in the art. For example, suitable lipid solubility can bedetermined by a partition test wherein a known quantity of the dye inorganic solvent is combined with the solvent or solvent system used inthe assay. If a partition results and, under the conditions used in theassay, there is no appreciable crossing by the dye into the solvent orsolvent system, then the dye is sufficiently lipid soluble. Put anotherway, the lipid soluble rhodamine dye should be sufficiently lipidsoluble such that it is capable of being imbibed into the polymer whendissolved in an organic solvent or solvent system and, when the dyedpolymer is subjected to the aqueous conditions of the assay, the dyeshould resist leaching out of the polymer to any degree thatsignificantly impacts the fluorescent signature of the dye imbibedpolymer or the results of the assay.

Generally, useful lipid-soluble rhodamines are rhodamines that aresubstituted at one or both of the exocyclic amine and/or imminiumnitrogens with a lipophilic substituent group designed to impart theresultant rhodamine dye with lipophilic characteristics or properties.Thus, useful lipid-soluble rhodamines may include one or two lipophilicsubstituents at the exocyclic amine nitrogen and/or one or twolipophilic substituents at the exocyclic imminium nitrogen. Thelipophilic substituents, whether attached to the same or differentnitrogen atoms, may be the same or different. In one embodiment, thelipid-soluble rhodamine is a rhodamine in which the exocyclic amine andexocyclic imminium nitrogens are each substituted with identicallipophilic groups.

Lipid-soluble rhodamines may include lipophilic substitutents at otherpositions, as well. In certain embodiments, it may be desirable to“mask” polar substituents on a particular rhodamine with lipophilicsubstituent groups to alter the lipid-solubility of the resultantrhodamine. For example, rhodamines which include a C-9 phenylsubstituted at the ortho position with a carboxylate or sulfonate groupmay be reacted with a lipophilic alcohol or amine, such as an alkyl,aryl or arylalkyl alcohol or amine, to yield an ortho ester or amidethat contributes to the net lipophilic properties of the resultantrhodamine dye.

Lipophilic substituents are groups that impart the resultant rhodaminedye with lipophilic character or properties as denoted above. The natureof each lipophilic substituent is not critical, as long as the resultantrhodamine dye is lipid soluble. Non-limiting examples of suitablelipophilic substituents include unsubstitued (C4-C20) alkyls, (C5-C40)aryls, and (C6-C40) arylalkyls. Depending on the number of methylene andmethine units in the lipophilic substituent, the lipophilic substituentmay also include pendant or internal hydrophilic groups. For example, alipophilic substitutent may include one or more internal heteroatoms,such as one or more internal O, S, N or NH groups. As another example, alipophilic substituent may include one or more pendant polar orhydrophilic substituents, such as one or more pendant halogen, —OH, —SH,—NH₂, —C(O)OH, —C(O)NH₂ or other polar or hydrophilic groups. Thus,lipophilic substituents may also include substituted (C4-C20) alkyl,substituted (C5-C40) aryls and substituted (C6-C40) arylalkyls, as wellas substituted and unsubstituted (C4-C20) heteroalkyl, substituted andunsubstituted (C5-C40) heteroaryls and substituted and unsubstituted(C6-C40) arylalkyls. As a specific example, if the rhodamine contains aC-9 phenyl ring, (C10-20) alkyl esters and alkyl amides are oftenemployed in one or both of the positions ortho to the point of thephenyl ring's attachment. The number of internal or pendant polar orhydrophilic groups that may be included in a lipophilic substituent willdepend upon, among other factors, the number of methylene or methinegroups included in the lipophilic substitutent and the number oflipophilic substituents on the rhodamine dye. The nature and number oflipophilic groups necessary to make a rhodamine lipid soluble can varyfrom molecule to molecule, and will be apparent to those of skill in theart.

In one embodiment, lipid-soluble rhodamine dyes useful in thefluorescent polymeric materials of the invention comprise one of thefollowing “core” structures:

where R¹ R², R^(2′), R^(3′), R^(3″), R⁴, R^(4′), R^(5′), R⁵, R^(6″),R^(6′), R^(7′), R⁷, R⁸, R⁹, R¹¹ R¹², R¹³, R¹⁴ and R¹⁵ are selected fromhydrogen or the same or different substituent groups; at least one ofR^(3′), R^(3″), R^(6″) and R^(6′) is a lipophilic substituent; and R⁹ instructure (II) is a nonaromatic substituent. In one embodiment, all ofthe substituents, whether they represent R², R^(2′), R^(3′), R^(3″), R⁴,R^(4′), R^(5′), R⁵, R^(6″), R^(6′), R^(7′), R⁷, R⁸, R⁹, R¹¹, R¹², R³,R¹⁴ or R¹⁵, contain no more than 40 atoms. In another embodiment all ofthe substituents, whether they represent R², R^(2′), R^(3′), R^(3″), R⁴,R^(4′), R^(5′), R⁵, R^(6″)R^(6′), R^(7′), R⁷, R⁸, R⁹, R¹¹, R¹², R¹³, R¹⁴or R¹⁵, contain no more than 25 atoms.

Lipid solubility is imparted to the rhodamine dyes of structures (I),(II), (III) and (IV) through the combination of substituents selected.Lipophilic substituents at of R^(3′), R^(3″), R^(6″) and R^(6′) areespecially useful in this regard. In one embodiment, at least oneR^(3′), R^(3″), R^(6″) and R^(6′) is lipophilic. In another embodiment,at least two of R^(3′), R^(3″), R^(6″) and R^(6′) are lipophilicsubstituents. In yet another embodiment, all four of R^(3′), R^(3″),R^(6″) and R^(6′) are lipophilic substituents. These lipophilicsubstituents may be the same or different. Non-limiting examples ofsuitable lipophilic substituents include unsubstituted (C4-C20) alkyls,(C5-C40) aryls, and (C6-C40) arylalkyls. Depending on the number ofmethylene and methine units in the lipophilic substituent, thelipophilic substituent may also include pendant or internal hydrophilicgroups. For example, a lipophilic substitutent may include one or moreinternal heteroatoms, such as one or more internal O, S, N or NH groups.As another example, a lipophilic substituent may include one or morependant polar or hydrophilic substituents, such as one or more pendanthalogen, —OH, —SH, —NH₂, —C(O)OH, —C(O)NH₂ or other polar or hydrophilicgroups. Thus, lipophilic substituents may also include substituted(C4-C20) alkyl, substituted (C5-C40) aryls and substituted (C6-C40)arylalkyls, as well as substituted and unsubstituted (C4-C20)heteroalkyl, substituted and unsubstituted (C5-C40) heteroaryls andsubstituted and unsubstituted (C6-C40) arylalkyls.

The fluorescent properties of the lipid soluble rhodamine dyes can betuned by the selection of different substituents. as is well known inthe art. Especially beneficial substituents for tuning the lipid solublerhodamine dyes include the moieties on the 9-carbon, namely, R⁹, R¹¹,R¹², R¹³, R¹⁴ and R¹⁵. The R², R^(2′), R⁴, R^(4′) R^(5′), R⁵, R^(7′) andR⁷ substituents are also beneficial toward tuning the spectralproperties of the dyes.

Symmetry is an important factor in selecting optimal dyes. Accordingly,it is desirable, but not necessary, for the lipid soluble rhodamine dyesto have identical R^(3′) and R^(6′) substituents and/or identical R^(3″)and R^(6″) substituents and/or identical R⁴ and R⁵ substituents.Similarly, it is desirable, but not necessary, for the dyes to haveidentical R¹ and R⁸ substituents and/or identical R² and R⁷substituents. Finally, with respect to structure (II), it is desirable,but not necessary, for the benzo, naphtho or polycyclic aryleno groupsthat bridge R¹ and R² to be identical to the benzo, naphtho orpolycyclic aryleno groups that bridge R⁷ and R⁸. The presence of one ormore, and especially all, of these symmetries facilitates the productionof a strong emission spectrum with a narrow full width half max.

Typically, R¹ and R⁸ in structures (I), (III) and (IV) are not,simultaneously, pendant or fused benzo, naphtho or polycyclic arylenogroups. The simultaneous presence of two relatively rigid aromatic ringsimmediately next to the 9-carbon may generate steric hinderances whenthe C-9 position is substituted with a substituted or unsubstitutedphenyl substituent. Such steric hinderance is not a concern forstructure (II), where R⁹ is, by definition, a nonaromatic substituent.

Often, with respect to structures (I), (III) and (IV), the dyes are morelimited if the polymer utilized is neither activated nor in particulateform. Specifically, in such scenarios, if R^(3″) and R^(6″) are eachphenyl or substituted phenyl and if R^(3′) and R⁶ are each hydrogen,then at least two of R¹¹, R¹², R¹³, R¹⁴ and R¹⁵ must be other thanhydrogen.

It has been discovered that certain types of C-9 phenyl substitutedlipid soluble rhodamines have especially good fluorescent properties.These lipid soluble rhodamines depart from known C-9 phenyl substitutedrhodamines in many ways, including the fact that they do not possess anortho carboxylic acid or an ortho sulfonic acid moiety, or an amide,acid halide or salt derivative thereof. Often these lipid solublerhodamines have symmetrical substitutions on the C-9 phenyl ring. In oneembodiment, these lipid soluble rhodamine dyes comprise one of corestructures (I), (III), and (IV) above where at least one of R¹¹ and R¹⁵are selected, independently of one another, from (C1-C20) alkyl orheteroalkyl, (C1-C20) alkoxy, halo, (C1-C20) haloalkyl, amino, mercapto,(C1-C20) alkylthio, cyano, isocyano, cyanato, mercaptocyanato, nitroso,nitro, azido, sulfeno, sulfinyl, and sulfino, and the remainder of R¹²,R¹³ and R¹⁴ are selected, independently of one another, from hydrogen,(C1-C20) alkyl or heteroalkyl, (C1-C20) alkoxy, halo, (C1-C20)haloalkyl, amino, mercapto, (C1-C20) alkylthio, cyano, isocyano,cyanato, mercaptocyanato, nitroso, nitro, azido, sulfeno, sulfinyl, andsulfino. Alternatively, R¹¹ and R¹⁵ are each the same substituentselected from (C1-C20) alkyl or heteroalkyl, (C1-C20) alkoxy, halo,(C1-C20) haloalkyl, amino, mercapto, (C1-C20) alkylthio, cyano,isocyano, cyanato, mercaptocyanato, nitroso, nitro, azido, sulfeno,sulfinyl, and sulfino, and the remainder of R¹², R¹³ and R¹⁴ areselected, independently of one another, from hydrogen, (C1-C20) alkyl orheteroalkyl, (C1-C20) alkoxy, halo, (C1-C20) haloalkyl, amino, mercapto,(C1-C20) alkylthio, cyano, isocyano, cyanato, mercaptocyanato, nitroso,nitro, azido, sulfeno, sulfinyl, and sulfino. In yet another embodiment,R¹¹ and R¹⁵ are each the same substituent as just described and R¹², R¹³and R¹⁴ are either unsubstituted or substituted in the same manner asR¹¹ and R¹⁵. In still another embodiment, R¹¹ and R¹⁵ are each the samehalo group, such as chlorine or fluorine, and R¹², R¹³ and R¹⁴ are eachhydrogen or the same halo group selected for R¹¹ and R¹⁵.

5.6 Structure (I) Dyes

In one embodiment, the lipid soluble rhodamine dyes comprise corestructure (I) and, additionally, the substituents therein are defined asfollows:

-   -   R¹ is selected from hydrogen, R^(x), (C1-C20) alkyl or        heteroalkyl optionally substituted with one or more of the same        or different R^(b) groups, (C5-C20) aryl or heteroaryl        optionally substituted with one or more of the same or different        R^(a) or suitable R^(b) groups and (C6-C40) arylalkyl or        heteroaryl alkyl optionally substituted with one or more of the        same or different R^(a) or suitable R^(b) groups, or,        alternatively, R¹ is taken together with R² form part of a        benzo, naptho or polycyclic aryleno group which is optionally        substituted with one or more of the same or different R^(a) or        suitable R^(b) groups;    -   R² is selected from hydrogen, R^(x), (C1-C20) alkyl or        heteroalkyl optionally substituted with one or more of the same        or different R^(b) groups, (C5-C20) aryl or heteroaryl        optionally substituted with one or more of the same or different        R^(a) or suitable R^(b) groups and (C6-C40) arylalkyl or        heteroaryl alkyl optionally substituted with one or more of the        same or different R^(a) or suitable R^(b) groups, or,        alternatively, R² is taken together with R¹ to form part of a        benzo, naptho or polycyclic aryleno group which is optionally        substituted with one or more of the same or different R^(a) or        suitable R^(b) groups, or, alternatively, R² is taken together        with R^(3′) to form a 5- or 6-membered ring which is optionally        substituted with one or more of the same or different R^(a) or        suitable R^(b) groups;    -   R^(3′) is selected from hydrogen, (C4-C20) alkyl or heteroalkyl        optionally substituted with one or more of the same or different        R^(b) groups, (C5-C20) aryl or heteroaryl optionally substituted        with one or more of the same or different R^(a) or suitable        R^(b) groups and (C6-C40) arylalkyl or heteroarylalkyl        optionally substituted with one or more of the same or different        R^(a) or suitable R^(b) groups, or, alternatively, R^(3′) is        taken together with R² to form a 5- or 6-membered ring which is        optionally substituted with one or more of the same or different        R^(a) or suitable R^(b) groups;    -   R^(3″) is selected from (C4-C20) alkyl or heteroalkyl optionally        substituted with one or more of the same or different R^(b)        groups, (C5-C20) aryl or heteroaryl optionally substituted with        one or more of the same or different R^(a) or suitable R^(b)        groups and (C6-C40) arylalkyl or heteroaryl alkyl optionally        substituted with one or more of the same or different R^(a) or        suitable R^(b) groups, or, alternatively, R^(3″) is taken        together with R⁴ to form a 5- or 6-membered ring which is        optionally substituted with one or more of the same or different        R^(a) or suitable R^(b) groups;    -   R⁴ is selected from hydrogen, R^(x), (C1-C20) alkyl or        heteroalkyl optionally substituted with one or more of the same        or different R^(b) groups, (C5-C20) aryl or heteroaryl        optionally substituted with one or more of the same or different        R^(a) or suitable R^(b) groups and (C6-C40) arylalkyl or        heteroarylalkyl optionally substituted with one or more of the        same or different R^(a) or suitable R^(b) groups, or,        alternatively R⁴ is taken together with R^(3″) to form a 5- or        6-membered ring which is optionally substituted with one or more        of the same or different R^(a) or suitable R^(b) groups;    -   R⁵ is selected from hydrogen, R^(x), (C1-C20) alkyl or        heteroalkyl optionally substituted with one or more of the same        or different R^(b) groups, (C5-C20) aryl or heteroaryl        optionally substituted with one or more of the same or different        R^(a) or suitable R^(b) groups and (C6-C40) arylalkyl or        heteroarylalkyl optionally substituted with one or more of the        same or different R^(a) or suitable R^(b) group or,        alternatively R⁵ is taken together with R^(6″) to form a 5- or        6-membered ring which is optionally substituted with one or more        of the same or different R^(a) or suitable R^(b) groups;    -   R^(6″) is selected from (C4-C20) alkyl or heteroalkyl optionally        substituted with one or more of the same or different R^(b)        groups, (C5-C20) aryl or heteroaryl optionally substituted with        one or more of the same or different R^(a) or suitable R^(b)        groups and (C6-C40) arylalkyl or heteroarylalkyl optionally        substituted with one or more of the same or different R^(a) or        suitable R^(b) groups or, alternatively R^(6″) is taken together        with R⁵ to form a 5- or 6-membered ring which is optionally        substituted with one or more of the same or different R^(a) or        suitable R^(b) groups;    -   R^(6′) is selected from hydrogen, (C4-C20) alkyl or heteroalkyl        optionally substituted with one or more of the same or different        R^(b) groups, (C5-C20) aryl or heteroaryl optionally substituted        with one or more of the same or different R^(a) or suitable        R^(b) groups and (C6-C40) arylalkyl or heteroarylalkyl        optionally substituted with one or more of the same or different        R^(a) or suitable R^(b) groups, or, alternatively, R^(6′) is        taken together with R⁷ to form a 5- or 6-membered ring        optionally substituted with one or more of the same or different        R^(a) or suitable R^(b) groups;    -   R⁷ is selected from hydrogen, R^(x), (C1-C20) alkyl or        heteroalkyl optionally substituted with one or more of the same        or different R^(b) groups, (C5-C20) aryl or heteroaryl        optionally substituted with one or more of the same or different        R^(a) or suitable R^(b) groups and (C6-C40) arylalkyl or        heteroaryl alkyl optionally substituted with one or more of the        same or different R^(a) or R^(b) groups, or, alternatively, R⁷        is taken together with R^(6′) to form a 5- or 6-membered ring        optionally substituted with one or more of the same or different        R^(a) or suitable R^(b) groups, or, alternatively, R⁷ is taken        together with R⁸ to form part of a benzo, naptho or polycyclic        aryleno group which is optionally substituted with one or more        of the same or different R^(a) or suitable R^(b) groups;    -   R⁸ is selected from hydrogen, R^(x), (C1-C20) alkyl or        heteroalkyl optionally substituted with one or more of the same        or different R^(b) groups, (C5-C20) aryl or heteroaryl        optionally substituted with one or more of the same or different        R^(a) or suitable R^(b) groups and (C6-C40) arylalkyl or        heteroaryl alkyl optionally substituted with one or more of the        same or different R^(a) or suitable R^(b) groups, or,        alternatively, R⁸ together with R⁷ form part of a benzo, naptho        or polycyclic aryleno group which is optionally substituted with        one or more of the same or different R^(a) or suitable R^(b)        groups;    -   R¹¹ and R¹⁵ are each, independently of one another, selected        from hydrogen, halo, (C1-C20) alkyl, haloalkyl, —OR^(y),        —SR^(y), —SOR^(y), —SO₂R^(y), —C(O)OR^(y), —S(O)₂OR^(y), amide,        sulfonamide and nitrile;    -   R¹², R¹³ and R¹⁴ are each, independently of one another,        selected from hydrogen, R^(x), (C1-C20) alkyl or heteroalkyl        optionally substituted with one or more of the same or different        R^(b) groups, (C5-C20) aryl or heteroaryl optionally substituted        with one or more of the same or different R^(a) or suitable        R^(b) groups and (C6-C40) arylalkyl or heteroarylalkyl        optionally substituted with one or more of the same or different        R^(a) or suitable R^(b) groups;    -   R^(x) is selected from —NR^(c)R^(c), —OR^(d), —SR^(d), halo,        haloalkyl, —CN, —NC, —OCN, —SCN, —NO, —NO₂, —N₃, —S(O)R^(d),        —S(O)₂R^(d), —S(O)₂OR^(d), —S(O)NR^(c)R^(c), —S(O)₂NR^(c)R^(c),        —OS(O)R^(d) —OS(O)₂R^(d), —OS(O)₂OR^(d), —OS(O)₂NR^(c)R^(c),        —C(O)R^(d), —C(O)OR^(d), —C(O)NR^(c)R^(c), —C(NH)NR^(c)R^(c),        —OC(O)R^(d), —OC(O)OR^(d), —OC(O)NR^(c)R^(c) and        —OC(NH)NR^(c)R^(c);    -   R^(y) is selected from (C1-C20) alkyls or heteroalkyls        optionally substituted with lipophilic substituents, (C5-C20)        aryls or heteroaryls optionally substituted with lipophilic        substituents and (C6-C26) arylalkyl or heteroarylalkyls        optionally substituted with lipophilic substituents;    -   R^(a) is selected from hydrogen, (C1-C8) alkyl or heteroalkyl,        (C5-C20) aryl or heteroaryl and (C6-C28) arylalkyl or        heteroarylalkyl;    -   R^(b) is selected from —NR^(c)R^(c), ═O, —OR^(d) ═S, —SR^(d),        ═NR^(d), ═NOR^(d), halo, haloalkyl, —CN, —NC, —OCN, —SCN, —NO,        —NO₂, ═N₂, —N₃, —S(O)R^(d), S(O)₂R^(d), —S(O)₂OR^(d),        —S(O)NR^(c)R^(c), —S(O)₂NR^(c)R^(c), —OS(O)R^(d), —OS(O)₂R^(d),        —OS(O)₂OR^(d), —OS(O)₂NR^(c)R^(c), —C(O)R^(d), —C(O)OR^(d),        —C(O)NR^(c)R^(c), —C(NH)NR^(c)R^(c), —OC(O)R^(d), —OC(O)OR^(d),        OC(O)NR^(c)R^(c) and —OC(NH)NR^(c)R^(c);    -   each R^(c) is independently hydrogen or R^(d), or,        alternatively, each R^(c) is taken together with the nitrogen        atom to which it is bonded to form a 5 to 8-membered saturated        or unsaturated ring which may optionally include one or more of        the same or different additional heteroatoms and which may        optionally be substituted with one or more of the same or        different R^(a) or R^(d) groups;    -   each R^(d) is independently R^(a) or R^(a) substituted with one        or more of the same or different R^(a) or R^(e) groups;    -   each R^(e) is selected from —NR^(a)R^(a), ═O, OR^(a), ═S,        —SR^(a), ═NR^(a), ═NOR^(a), halo, haloalkyl, —CN, —NC, —OCN,        —SCN, —NO, —NO₂, ═N₂, —N₃, —S(O)R^(a), —S(O)₂R^(a),        —S(O)₂OR^(a), S(O)NR^(a)R^(a), —S(O)₂NR^(a)R^(a), —OS(O)R^(a),        —OS(O)₂R^(a), —OS(O)₂OR^(a), —OS(O)₂NR^(a)R^(a), —C(O)R^(a),        —C(O)OR^(a), —C(O)NR^(a)R^(a), —C(NH)NR^(a)R^(a), —OC(O)R^(a),        —OC(O)OR^(a), —OC(O)NR^(a)R^(a) and —OC(NH)NR^(a)R^(a).

In another embodiment, the lipid soluble rhodamine dyes comprising corestructure (I) are as described in U.S. Pat. Nos. 5,231,191, 5,366,860,5,410,053, 5,750,409, 5,840,999, 5,847,162, 5,936,087, 6,008,379 and6,248,884 with the exception that at least one, and as many as all, ofR^(3′), R^(3″), R^(6′) and R^(6″) are selected from lipophilic moieties.Accordingly, these patents are hereby incorporated by reference. Onceagain, non-limiting examples of lipophilic moieties include (C4-C20)alkyls, (C5-C20) aryls, and (C6-C40) arylalkyls.

In another embodiment, the lipid soluble rhodamine dyes comprise corestructure (I) and, additionally, the substituents therein comprise oneor more of the following features: R¹ and R⁸ are each, independently ofone another, selected from hydrogen, (C1-C8) alkyl, halo, and —CF₃, orare part of a fused aryl or heteroaryl group with R² and R⁷,respectively; R² and R⁷ are each, independently of one another, selectedfrom hydrogen, (C1-C8) alkyl, halo, —CF₃, —CN, carboxylate, ester,sulfone, amino, amido and oxoether, or are part of a fused aryl orheteroaryl group with R¹ and R⁸, respectively, or are part of a 5 or 6membered ring with R^(3′) and R^(6′), respectively; R^(3′) and R^(6′)are each, independently of one another, selected from hydrogen and(C4-C20) alkyl, or are part of a 5 or 6 membered ring with R² and R⁷,respectively; R^(3″) and R^(6″) are each, independently of one another,selected from hydrogen and (C4-C20) alkyl or are part of a 5 or 6membered ring with R⁴ and R⁵, respectively; at least two of R^(3′),R^(3″), R^(6′), R^(6″) are not hydrogen; R⁴ and R⁵ are each,independently of one another, selected from hydrogen, (C1-C8) alkyl,halo, —CF₃, aryl, heteroaryl, —CN, carboxylate, ester, and sulfone, orare part of a 5 or 6 membered ring with R^(3″) and R^(6″), respectively;R¹¹ and R¹⁵ are each, independently, selected from hydrogen, halo,ester, amide, sulfonamide, (C1-C20) alkyl, —OR^(e), —SR^(e), —CN andsulfone; and R¹², R¹³ and R¹⁴ are each, independently of one another,selected from hydrogen, halo, alkyl, aryl, (C4 to C20) alkyl ester,sulfone, —CF₃ and —CN.

In yet another embodiment, the lipid soluble rhodamine dyes comprisecore structure (I) and, additionally, the substituents therein compriseone or more of the following features: R¹ and R⁸ are each, independentlyof one another, selected from hydrogen, (C1-C8) alkyl, and halo, or arepart of a fused aryl or heteroaryl group with R² and R⁸, respectively;R² and R⁷ are each, independently of one another, selected fromhydrogen, (C1-C8) alkyl, and halo, or are part of a fused aryl orheteroaryl group with R¹ and R⁸ respectively, or are part of a 5 or 6membered ring with R^(3′) and R^(6′), respectively; R^(3′) and R^(6′)are each the same or different (C4-C20) alkyl; R^(3″) and R^(6″) areeach, independently of one another, selected from hydrogen and the sameor different (C4-C20) alkyl, or are part of a 5 or 6 membered ring withR⁴ and R⁵, respectively; R⁴ and R⁵ are each, independently of oneanother, selected from hydrogen, (C1-C8) alkyl, aryl and heteroaryl andhalo, or are part of a 5 or 6 membered ring with R^(3″) and R^(6″),respectively; R¹¹ and R¹⁵, independently of one another, selected fromhydrogen, fluoro, chloro, (C1-C20) alkyl, —OR^(e) and —SR^(e); and R¹²,R¹³ and R¹⁴ are each, independently of one another, selected fromhydrogen and halo.

In still another embodiment, the lipid soluble rhodamine dyes compriseone of the following structural embodiments of core structure (I):

wherein each R^(z) is independently selected from hydrogen, R^(b),(C1-C20) alkyl optionally substituted with one or more of the same ordifferent R^(b) groups, (C5-C20) aryl optionally substituted with one ormore of the same or different R^(a) or suitable R^(b) groups and(C6-C40) arylalkyl optionally substituted with one or more of the sameor different R^(a) or suitable R^(b) group; and R¹, R², R^(3′), R^(3″),R⁴, R⁵, R^(6″), R⁶, R⁷, R⁸, R¹¹, R¹²R¹³, R¹⁴, R¹⁵, R^(a) and R^(b) areas previously defined. Often, R^(Z) is selected from hydrogen and(C1-C8) alkyl.

The following lipid soluble rhodamine dyes are non-limiting examples ofdyes that comprise core structure (I):

5.7 Structure (II) Dyes

In one embodiment, the lipid soluble rhodamine dyes comprise corestructure (II) and, additionally, the substituents therein are definedas follows:

-   -   R¹, R², R^(3′), R^(3″), R⁴, R⁵, R^(6″), R^(6′), R⁷, R⁸, R^(x),        R^(a), R^(b), R^(e), R^(d), and R^(e) are as first defined in        section 5.6 with respect to core structure (I); and    -   R⁹ is selected from hydrogen, alkyl, halo, haloalkyl, or        nitrile.

In another embodiment, extended rhodamines comprising core structure(II) are as described in U.S. Pat. Nos. 6,008,379 and 5,936,087, withthe exception that at least one, and as many as all, of R^(3′), R^(3″),R^(6′) and R^(6″) are selected from lipophilic moieties. Accordingly,these patents are hereby incorporated by reference. Furthermore, theserhodamines can be extended as previously discussed and illustrated inU.S. Pat. No. 6,248,884, which is also incorporated herein by reference.Non-limiting examples of lipophilic moieties include (C4-C20) alkyls,(C5-C20) aryls and (C6-C40) arylalkyls.

In another embodiment, the lipid soluble rhodamine dyes comprise corestructure (II) and the substituents therein comprise one or more of thefollowing features: R¹ when taken together with R² forms part of a fusedbenzo, naptho or polycyclic aryleno group that is optionally substitutedwith one or more of the same or different R^(a) or suitable R^(b)groups; R⁷ when taken together with R⁸ form part of a fused benzo,naptho or polycyclic aryleno group that is optionally substituted withone or more of the same or different R^(a) or suitable R^(b) groups; andR⁹ is hydrogen.

In yet another embodiment, the lipid soluble rhodamine dyes comprise oneof the following structural embodiments of core structure (II):

wherein each R^(Q) is independently selected from hydrogen, (C1-C20)alkyl optionally substituted with one or more of the same or differentR^(b) groups, (C5-C20) aryl optionally substituted with one or more ofthe same or different R^(a) or suitable R^(b) groups and (C6-C40)arylalkyl optionally substituted with one or more of the same ordifferent R^(a) or suitable R^(b) group; and R^(a) and R^(b) are aspreviously defined. Preferably, each R^(Q) is independently selectedfrom the group consisting of hydrogen and (C1-C8) alkyl.

The following lipid soluble rhodamine dyes are non-limiting examples ofspecific dyes that comprise core structure (II):

5.8 Structure (III) Dyes

In one embodiment, the lipid soluble rhodamine dyes comprise corestructure (III) and, additionally, the substituents therein are definedas follows:

-   -   R¹, R^(3″), R⁵, R^(6″), R^(6′), R⁷, R⁸, R¹¹, R¹², R¹³, R¹⁴, R¹⁵        R^(x), R^(y), R^(a), R^(b), R^(c), R^(d), and R^(e) are as first        defined in section 5.6 with respect to core structure (I);    -   R² is selected from hydrogen, R^(x), (C1-C20) alkyl or        heteroalkyl optionally substituted with one or more of the same        or different R^(b) groups, (C5-C20) aryl or heteroaryl        optionally substituted with one or more of the same or different        R^(a) or suitable R^(b) groups and (C6-C40) arylalkyl or        heteroaryl alkyl optionally substituted with one or more of the        same or different R^(a) or suitable R^(b) groups, or,        alternatively, R² is taken together with R¹ or R² to form part        of a benzo, naptho or polycyclic aryleno group which is        optionally substituted with one or more of the same or different        R^(a) or suitable R^(b) groups;    -   R^(2′) is selected from hydrogen, R^(x), (C1-C20) alkyl or        heteroalkyl optionally substituted with one or more of the same        or different R^(b) groups, (C5-C20) aryl or heteroaryl        optionally substituted with one or more of the same or different        R^(a) or suitable R^(b) groups and (C6-C40) arylalkyl or        heteroaryl alkyl optionally substituted with one or more of the        same or different R^(a) or suitable R^(b) groups, or,        alternatively, R^(2′) is taken together with R² to form part of        a benzo, naptho or polycyclic aryleno group which is optionally        substituted with one or more of the same or different R^(a) or        suitable R^(b) groups, or, alternatively, R^(2′) is taken        together with R^(3′) to form a 5- or 6-membered ring which is        optionally substituted with one or more of the same or different        R^(a) or suitable R^(b) groups;    -   R^(3′) is selected from hydrogen, (C4-C20) alkyl or heteroalkyl        optionally substituted with one or more of the same or different        R^(b) groups, (C5-C20) aryl or heteroaryl optionally substituted        with one or more of the same or different R^(a) or suitable        R^(b) groups and (C6-C40) arylalkyl or heteroarylalkyl        optionally substituted with one or more of the same or different        R^(a) or suitable R^(b) groups, or, alternatively, R^(3′) is        taken together with R^(2′) to form a 5- or 6-membered ring which        is optionally substituted with one or more of the same or        different R^(a) or suitable R^(b) groups;    -   R⁴ is selected from hydrogen, R^(x), (C1-C20) alkyl or        heteroalkyl optionally substituted with one or more of the same        or different R^(b) groups, (C5-C20) aryl or heteroaryl        optionally substituted with one or more of the same or different        R^(a) or suitable R^(b) groups and (C6-C40) arylalkyl or        heteroarylalkyl optionally substituted with one or more of the        same or different R^(a) or suitable R^(b) groups, or,        alternatively R⁴ is taken together with R^(3″) to form a 5- or        6-membered ring which is optionally substituted with one or more        of the same or different R^(a) or suitable R^(b) groups, or,        alternatively, R⁴ is taken together with R^(4′) to form part of        a benzo, naptho or polycyclic aryleno group which is optionally        substituted with one or more of the same or different R^(a) or        suitable R^(b) groups; and    -   R^(4′) is selected from hydrogen, R^(x), (C1-C20) alkyl or        heteroalkyl optionally substituted with one or more of the same        or different R^(b) groups, (C5-C20) aryl or heteroaryl        optionally substituted with one or more of the same or different        R^(a) or suitable R^(b) groups and (C6-C40) arylalkyl or        heteroarylalkyl optionally substituted with one or more of the        same or different R^(a) or suitable R^(b) groups, or,        alternatively R^(4′) is taken together R⁴ to form part of a        benzo, naptho or polycyclic aryleno group which is optionally        substituted with one or more of the same or different R^(a) or        suitable R^(b) groups.

In another embodiment, extended rhodamines comprising core structure(III) are as described in U.S. Pat. No. 6,248,884 with the exceptionthat at least one, and as many as all, of R^(3′), R^(3″), R^(6′) andR^(6″) are selected from lipophilic moieties. Accordingly, U.S. Pat. No.6,248,884 is hereby incorporated by reference. Non-limiting examples oflipophilic moieties include (C4-C20) alkyls, (C5-C20) aryls, and(C6-C40) arylalkyls.

In yet another embodiment, the extended rhodamines comprising corestructure (III) additionally comprises one or more substituted orunsubstituted five or six membered fused rings that comprise, as part ofthe ring, a nitrogen atom from either the exocyclic amine or exocyclicimminium group and one of R^(2′), R⁴, R⁵ or R⁷. For example, R^(2′) canform a fused substituted or unsubstituted five or six membered ring withR^(3′). Alternatively, R⁴ can form a fused substituted or unsubstitutedfive or six membered ring with R^(3″). Alternatively, R⁵ can form afused substituted or unsubstituted ring with R^(6″). Alternatively, R⁷can form a fused substituted or unsubstituted ring with R^(6′). Oftentwo fused rings are present, one that includes the imminium nitrogen andone that includes the amine nitrogen. Illustrative substituent groups onthe five or six membered rings include R^(a) or suitable R^(b) groups.In one embodiment the substituents on the five or six membered ringsinclude (C7-C20) alkyls, phenyl or alkylphenyl groups, and (C7-C20)alkylester substituted phenyl or alkylphenyl groups.

5.9 Structure (IV) Dyes

In one embodiment, the lipid soluble rhodamine dyes comprise corestructure (IV) and, additionally, the substituents therein are definedas follows:

-   -   R¹, R^(3″), R^(6″), R⁸, R¹¹, R¹², R¹³, R¹⁴, R¹⁵ R^(x), R^(y),        R^(a), R^(b), R^(c), R^(d), and R^(e) are as first defined in        section 5.6 with respect to core structure (I);    -   R², R^(2′), R^(3′), R⁴, and R^(4′) are as defined in section 5.8        with respect to core structure (III);    -   R^(5′) is selected from hydrogen, R^(x), (C1-C20) alkyl or        heteroalkyl optionally substituted with one or more of the same        or different R^(b) groups, (C5-C20) aryl or heteroaryl        optionally substituted with one or more of the same or different        R^(a) or suitable R^(b) groups and (C6-C40) arylalkyl or        heteroarylalkyl optionally substituted with one or more of the        same or different R^(a) or suitable R^(b) group or,        alternatively R⁵ is taken together with R⁵ to form part of a        benzo, naptho or polycyclic aryleno group which is optionally        substituted with one or more of the same or different R^(a) or        suitable R^(b) groups.    -   R⁵ is selected from hydrogen, R^(x), (C1-C20) alkyl or        heteroalkyl optionally substituted with one or more of the same        or different R^(b) groups, (C5-C20) aryl or heteroaryl        optionally substituted with one or more of the same or different        R^(a) or suitable R^(b) groups and (C6-C40) arylalkyl or        heteroarylalkyl optionally substituted with one or more of the        same or different R^(a) or suitable R^(b) group or,        alternatively R⁵ is taken together with R^(6″) to form a 5- or        6-membered ring which is optionally substituted with one or more        of the same or different R^(a) or suitable R^(b) groups, or,        alternatively, R⁵ is taken together with R^(5′) to form part of        a benzo, naptho or polycyclic aryleno group which is optionally        substituted with one or more of the same or different R^(a) or        suitable R^(b) groups;    -   R^(6′) is selected from hydrogen, (C4-C20) alkyl or heteroalkyl        optionally substituted with one or more of the same or different        R^(b) groups, (C5-C20) aryl or heteroaryl optionally substituted        with one or more of the same or different R^(a) or suitable        R^(b) groups and (C6-C40) arylalkyl or heteroarylalkyl        optionally substituted with one or more of the same or different        R^(a) or suitable R^(b) groups, or, alternatively, R^(6′) is        taken together with R^(7′) to form a 5- or 6-membered ring        optionally substituted with one or more of the same or different        R^(a) or suitable R^(b) groups;    -   R^(7′) is selected from hydrogen, R^(x), (C1-C20) alkyl or        heteroalkyl optionally substituted with one or more of the same        or different R^(b) groups, (C5-C20) aryl or heteroaryl        optionally substituted with one or more of the same or different        R^(a) or suitable R^(b) groups and (C6-C40) arylalkyl or        heteroaryl alkyl optionally substituted with one or more of the        same or different R^(a) or R^(b) groups, or, alternatively,        R^(7′) is taken together with R^(6′) to form a 5- or 6-membered        ring optionally substituted with one or more of the same or        different R^(a) or suitable R^(b) groups, or, alternatively,        R^(7′) is taken together with R⁷ to form part of a benzo, naptho        or polycyclic aryleno group which is optionally substituted with        one or more of the same or different R^(a) or suitable R^(b)        groups; and

R⁷ is selected from hydrogen, R^(x), (C1-C20) alkyl or heteroalkyloptionally substituted with one or more of the same or different R^(b)groups, (C5-C20) aryl or heteroaryl optionally substituted with one ormore of the same or different R^(a) or suitable R^(b) groups and(C6-C40) arylalkyl or heteroaryl alkyl optionally substituted with oneor more of the same or different R^(a) or R^(b) groups, or,alternatively, R⁷ is taken together with R^(7′) or R⁸ to form part of abenzo, naptho or polycyclic aryleno group which is optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups.

In another embodiment, extended rhodamines corresponding to corestructure (IV) are as described in U.S. Pat. No. 6,248,884 with theexception that at least one, and as many as all, of R³, R^(3″), R^(6′)and R^(6″) are selected from lipophilic moieties. Accordingly, U.S. Pat.No. 6,248,884 is hereby incorporated by reference. Non-limiting examplesof lipophilic moieties include (C4-C20) alkyls, (C5-C20) aryls, and(C6-C40) arylalkyls.

In yet another embodiment, the extended rhodamines comprising corestructure (IV) additionally comprises one or more substituted orunsubstituted five or six membered fused rings that comprise, as part ofthe ring, a nitrogen atom from either the exocyclic amine or exocyclicimminium group and one of R^(2′), R⁴, R⁵ or R⁷. For example, R^(2′) canform a fused substituted or unsubstituted five or six membered ring withR^(3′). Alternatively, R⁴ can form a fused substituted or unsubstitutedfive or six membered ring with R^(3″). Alternatively, R⁵ can form afused substituted or unsubstituted ring with R^(6″). Alternatively,R^(7′) can form a fused substituted or unsubstituted ring with R^(6′).Often two fused rings are present, one that includes the imminiumnitrogen and one that includes the amine nitrogen. Illustrativesubstituent groups on the five or six membered rings include R^(a) orsuitable R^(b) groups. In one embodiment the substituents on the five orsix membered rings include (C7-C20) alkyls, phenyl or alkylphenylgroups, and (C7-C20) alkylester substituted phenyl or alkylphenylgroups.

5.10 Linking Groups

As discussed previously, in some embodiments, the lipid-solublerhodamine dye may be attached to the polymeric material by way of acovalent linkage. In such embodiments, the lipid-soluble rhodamine dyewill typically include a linking group suitable for forming the covalentlinkage. The identity of the linking group will depend upon thefunctional group of the polymer to which the lipid-soluble rhodamine dyewill be attached, and will be apparent to those of skill in the art. Inone embodiment, the linking group is a nucleophilic or electrophilicgroup capable of forming a covalent linkage with a complementaryelectrophilic or nucleophilic group on the polymer. Non-limitingexamples of suitable electrophilic linking groups include any one or acombination of the following: amines/anilines, alcohols/phenols, thiols,hydrazines and hydroxylamines. Non-limiting examples of suitableelectrophilic linking groups include any one or a combination of thefollowing: activated esters such as pentafluorophenyl ester andNHS-ester, acrylamides, acyl azides, acyl halides, acyl nitriles,aldehydes or ketones, alkyl halides, alkyl sulfonates, anhydrides, arylhalides, aziridines, boronates, carboxylic acids, carbodiimides,diazoalkenes, epoxides, haloacetamides, halotriazines, imido esters,isocyanates, isothiocyanates, maleimides, phosphoramidites, silylhalides, sulfonate esters and sulfonyl halides.

The linking group may be attached directly to the lipid-solublerhodamine or it may be spaced away from the rhodamine by way of aspacing moiety. The nature of the spacing moiety is not critical, andmay vary broadly. For example, the spacing moiety may be a substitutedor unsubstituted alkylene or heteroalkylene, a substituted orunsubstituted arylene or heteroarylene, a substituted or unsubstitutedarylalkylene or heteroarylalkylene, or a combination of such groups. Inone embodiment, the spacing moiety is an unsubstituted alkylene of theformula —(CH₂)_(n)—, where n is an integer ranging from 1 to 40,typically from 1 to 20 and more typically from 1 to 10. Other exemplaryspacing moieties suitable for spacing the linking group from theremainder of the lipid-soluble rhodamine molecule are described, forexample, in U.S. Pat. Nos. 5,863,727, 5,847,162, 6,229,055, 6,248,884,and 6,372,907. A lipid-soluble rhodamine may include one or a pluralityof linking groups, which may be the same or different.

In one embodiment, a lipid-soluble rhodamine suitable for covalentattachment to the polymer is any of the previously-describedlipid-soluble rhodamines in which one or R¹, R², R^(2′), R⁴, R^(4′), R⁵,R^(5′), R⁷, R^(7′), R⁸, R¹¹, R¹², R¹³, R¹⁴ or R¹⁵ is a substitutent ofthe formula —S-LG, where S represents a bond or a spacing moiety and LGrepresents a linking group. In a specific embodiment, one of R⁴, R⁵,R¹², R¹³ or R¹⁴ is —S-LG.

5.11 Energy Transfer Dyes

The lipid soluble rhodamine dyes may be part of an energy transfer(“ET”) network comprising, for example, two to four dyes covalentlyattached to one another that transfer energy to generate a longerStoke's shift. One example of an ET network would be a fluorescenceresonance energy transfer (“FRET”) dye. In other words, the lipidsoluble rhodamine dyes may be part of series of dyes that are covalentlyattached to one another wherein at least one of the dyes is a lipidsoluble rhodamine. Linkages for covalently attaching rhodamine dyes toother dyes are known in the art, as are suitable locations forattachment to the rhodamine dyes (see, e.g., U.S. Pat. Nos. 5,800,996and 5,863,727). In one embodiment, each dye in the energy transfernetwork is within 5 to 100 Å of the neighboring dye or dyes in thenetwork to which it is covalently attached. In such embodiments, thelipid soluble rhodamine dye can be the donor, acceptor, or anintermediate dye in the network.

Thus, in one embodiment, the lipid-soluble rhodamine dyes useful in thefluorescent polymeric materials of the invention include anylipid-soluble dye comprising one of the following “core” structures:

wherein where at least one of R³, R^(3″), R^(6″) and R^(6′) represents alipophilic substituent, R⁹ (present in core structure (II) only) is anonaromatic substituent, at least one of R², R^(2′), R^(3′), R⁴, R^(4′),R^(5′), R⁵, R^(6′), R^(7′), R⁷, R¹², R¹³, and R¹⁴ corresponds to themoiety —S¹-LK-S²-D, and the remainder of R¹, R², R^(2′), R^(3′), R^(3″),R⁴, R^(4′), R^(5′), R⁵, R^(6″), R^(6′), R^(7′), R⁷, R⁸, R⁹, R¹¹, R¹²,R¹³, R¹⁴ and R¹³ are, independently, selected from hydrogen or the sameor different substituent groups having no more than 40 atoms. In thisinstance, D represents a covalently attached acceptor or donor dye andthe moieties S¹, LK, and S² form what is known in the art as a“linker”—which embraces any functionality known in the art that attachesone dye to another.

The identity of donor or acceptor dye D is not critical, so long as itcan donate or accept energy from or to the particular lipid-solublerhodamine to which it is attached. Dyes that can act as donor oracceptors for rhodamines are well-known, and include, for example,fluoresceins, rhodamines, cyanines, phthalocyanine and squaraine dyes.Any of these dyes, or another lipid-soluble rhodamine as describedherein, may be used as the donor dye or acceptor dye in an energytransfer dye comprising a lipid-soluble rhodamine. The ability to selecta suitable dye D for a particular lipid-soluble rhodamine is within theroutine skill in the art.

S¹ and S² are, independently of one another, a covalent bond or aspacing moiety. Like the previously-described spacing moiety “S,” thenature of spacing moieties S¹ and S² may vary broadly, and may includesubstituted or unsubstituted alkylene or heteroalkylenes, substituted orunsubstituted arylenes or heteroarylene, or substituted or unsubstitutedarylalkylenes or heteroarylalkylenes, or combinations thereof.

LK represents a linkage, which may be a bond or another type of linkage,such as a linkage formed between a nucleophilic (or electrophilic) groupand a complementary electrophilic (or nucleophilic) group. In oneembodiment, LK is selected from an ester, an amide, a sulfonamide, ahydrazine, an imine, a maleimide, a sulfide, a disulfide, a carbamateand a thiocarbamate linkage.

As will be appreciated by skilled artisans, the various substitutentsS¹, LK and S of the linker should be selected to position thelipid-soluble rhodamine and acceptor or donor dye in close enoughproximity to one another such that the dyes can undergo energy transfer,whether via FRET or another mechanism.

Suitable linkers are illustrated, for example, by U.S. Pat. Nos.5,800,996 and 5,863,727, issued to Lee et al., U.S. Pat. No. 6,008,279,issued to Benson et al., and U.S. Pat. No. 5,654,419, issued to Mathieset al., all of which are hereby incorporated by reference. Methods ofsynthesizing such energy transfer dyes, as well as suitable points ofattachment for covalently coupling the lipid-soluble rhodamine andacceptor or donor dye D to one another are also described in thesepatents.

In one exemplary embodiment, an energy transfer dye of the formulaD¹-S¹-LK-S2-D, where D¹ represents a lipid-soluble rhodamine dye and S¹,LK, S2 and D are as defined above may be synthesized by reacting alipid-soluble rhodamine dye of the formula D¹-S¹-LG, where LG representsa linking group, with a donor or acceptor dye of the formula D-S²-LG′,where LG′ represents a linking group which is complementary to linkinggroup LG such that LG and LG′ may react with one another to form linkageLK. As a specific embodiment, LG may be an activated ester such as anNHS-ester and LG′ may be a primary amino group, such that reaction formsan amide linkage LK.

5.12 Dye Combinations

The lipid soluble rhodamine dyes may be used in combination with oneanother. The lipid soluble rhodamine dyes may also be used incombination with any other class of dye that is fluorescent and can beattached and/or absorbed into the polymer. In one embodiment, acombination of dyes is employed wherein all of the dyes fluoresce whenirradiated by the same excitation source. In another embodiment,multiple excitation sources are employed.

Nonlimiting examples of additional dyes that may be employed includeacridine dyes, alizarene dyes, azo dyes, anthraquinine dyes, bodipydyes, coumarin dyes, cyanine dyes, fluorescein dyes such as AMFAM andPET, lanthanide complexes, oxazine dyes, phenazathionium dyes,phenazoxonium dyes, porphyorin dyes, pyrene dyes, pyrilium dyes,perylene dyes, phenoxazine dyes, phenezine dyes, rhodol dyes andxanthene dyes. Other dyes that may be utilized include, but are notlimited to the following: squaraine derivatives; phthalocyanines;naphthalocyanines, 3-Hydroxypyrene 5,8,10-Tri Sulfonic acid, 5-HydroxyTryptamine, 5-Hydroxy Tryptamine (5-HT), Acid Fuhsin, Acridine Orange,Acridine Red, Acridine Yellow, Acriflavin, AFA (Acriflavin FeulgenSITSA), Alizarin Complexon, Alizarin Red, Allophycocyanin, ACMA,Aminoactinomycin D, Aminocoumarin, Anthroyl Stearate, Aryl- orHeteroaryl-substituted Polyolefin, Astrazon Brilliant Red 4G, AstrazonOrange R, Astrazon Red 6B, Astrazon Yellow 7 GLL, Atabrine, Auramine,Aurophosphine, Aurophosphine G, BAO 9 (Bisaminophenyloxadiazole), BCECF,Berberine Sulphate, Bisbenzamide, BOBO 1, Blancophor FFG Solution,Blancophor SV, Bodipy F1, BOPRO 1, Brilliant Sulphoflavin FF, CalcienBlue, Calcium Green, Calcofluor RW Solution, Calcofluor White, CalcophorWhite ABT Solution, Calcophor White Standard Solution, Carbocyanine,Carbostyryl, Cascade Blue, Cascade Yellow, Catecholamine, Chinacrine,Coriphosphine O, Coumarin, Coumarin-Phalloidin, CY3.1 8, CY5.1 8, CY7,Dans (1-Dimethyl Amino Naphaline 5 Sulphonic Acid), Dansa (DiaminoNaphtyl Sulphonic Acid), Dansyl NH—CH₃, DAPI, Diamino Phenyl Oxydiazole(DAO), Dimethylamino-5-Sulphonic acid, Dipyrrometheneboron Difluoride,Diphenyl Brilliant Flavine 7GFF, Dopamine, Eosin, Erythrosin ITC,Ethidium Bromide, Euchrysin, FIF (Formaldehyde Induced Fluorescence),Flazo Orange, Fluo 3, Fluorescamine, Fura-2, Genacryl Brilliant Red B,Genacryl Brilliant Yellow 10GF, Genacryl Pink 3G, Genacryl Yellow 5GF,Gloxalic Acid, Granular Blue, Haematoporphyrin, Hoechst 33258, Indo-1,Intrawhite Cf Liquid, Leucophor PAF, Leucophor SF, Leucophor WS,Lissamine Rhodamine B200 (RD200), Lucifer Yellow CH, Lucifer Yellow VS,Magdala Red, Marina Blue, Maxilon Brilliant Flavin 10 GFF, MaxilonBrilliant Flavin 8 GFF, MPS (Methyl Green Pyronine Stilbene),Mithramycin, NBD Amine, Nile Red, Nitrobenzoxadidole, Noradrenaline,Nuclear Fast Red, Nuclear Yellow, Nylosan Brilliant Flavin E8G, OregonGreen, Oxazine, Oxazole, Oxadiazole, Pacific Blue, Pararosaniline(Feulgen), Phorwite AR Solution, Phorwite BKL, Phorwite Rev, PhorwiteRPA, Phosphine 3R, Phthalocyanine, Phycoerythrin R, PolyazaindacenePontochrome Blue Black, Porphyrin, Primuline, Procion Yellow, PropidiumIodide, Pyronine, Pyronine B, Pyrozal Brilliant Flavin 7GF, QuinacrineMustard, Rhodamine 123, Rhodamine 5 GLD, Rhodamine 6G, Rhodamine B,Rhodamine B 200, Rhodamine B Extra, Rhodamine BB, Rhodamine BG,Rhodamine WT, Rose Bengal, Serotonin, Sevron Brilliant Red 2B, SevronBrilliant Red, 4G, Sevron Brilliant Red B, Sevron Orange, Sevron YellowL, SITS (Primuline), SITS (Stilbene Isothiosulphonic acid), Stilbene,Snarf 1, sulpho Rhodamine B Can C, Sulpho Rhodamine G Extra,Tetracycline, Texas Red, Thiazine Red R, Thioflavin S, Thioflavin TCN,Thioflavin 5, Thiolyte, Thiozol Orange, Tinopol CBS, TOTO 1, TOTO 3,True Blue, Ultralite, Uranine B, Uvitex SFC, Xylene Orange, XRITC, YOPRO1, or combinations thereof.

In one embodiment, a lipid soluble rhodamine dye is used in combinationwith a total of one to ten additional dyes which may include additionallipid soluble rhodamine dyes. When the dyes are not covalently attachedto the polymer component, it is beneficial for the dyes to be soluble acommon hydrophobic organic solvent to minimize the steps necessary toadequately imbibe the dyes into the polymer.

The fluorescent dyes are selected based on the desired emission,absorption and solubility properties. The spectral properties of thefluorescent dyes selected should be sufficiently close in excitationwavelengths and intensity to the lipid soluble rhodamine dyes to permitthe use of the same analytical equipment, e.g., flow cytometryequipment.

5.13 Methods of Dye Synthesis

The lipid-soluble rhodamine dyes may be synthesized by well-knownmethods or by routine modification of such methods. For example,lipid-soluble rhodamine dyes which include a C9-phenyl substitutenthaving an ortho carboxylate or sulfonate substituent may be prepared bythe methods described in U.S. Pat. No. 6,372,907, U.S. Pat. No.6,248,884, U.S. Pat. No. 6,020,481, U.S. Pat. No. 6,008,379, U.S. Pat.No. 5,847,162, U.S. Pat. No. 5,936,087, U.S. Pat. No. 5,847,162, U.S.Pat. No. 5,750,409, U.S. Pat. No. 5,410,053 and U.S. Pat. No. 5,231,191,the disclosures of which are incorporated herein by reference.Lipid-soluble rhodamine dyes which include an “extended rhodamine”parent ring, as exemplified by structures (III) and (IV) may be preparedby the methods described in U.S. Pat. No. 6,248,884 and U.S. Pat. No.6,008,379, the disclosures of which are incorporated herein byreference. Lipid-soluble rhodamine dyes which include a non-phenyl C9substitutent, as exemplified by structure (II), may be prepared by themethods described in U.S. Pat. No. 5,936,087 and U.S. Pat. No.6,248,884, the disclosures of which are incorporated herein byreference. Lipid-soluble rhodamine dyes which include a C9-phenylsubstitute which does not have an ortho carboxylate or sulfonatesubstituent may be prepared by the methods illustrated in FIGS. 1, 2 and3 as well as the methods described in copending patent application Ser.No. ______, entitled “Phenyl Xanthene Dyes,” filed concurrentlyherewith, the disclosure of which is incorporated herein by reference.Additional methods are provided in the Examples section.

5.14 Methods of Dye In Corporation

The method of incorporating the dye or dye mixture into or onto thepolymer depends on a number of factors, including the desiredcharacteristics of the fluorescent polymeric material, the intendedmethod and environment for using the fluorescent polymeric material,economic considerations, availability of certain items, etc. Thefluorescent dyes can be incorporated into the polymer by any methodknown in the art including, without limitation: (i) suspending the dyesand polymer in miscible solvent; and (ii) adding polymer to an organicsolvent that swells, but does not dissolve, the polymer, then adding dyein organic solvent, and then shrinking the dye filled polymers with apolar solvent such as methanol.

In one embodiment, the dyes are incorporated internally into polymermicroparticles, typically without covalent attachment, by absorption ofthe dyes into the polymeric microparticles while the dyes are dissolvedin an organic solvent. This offers the great advantage of being able toprepare uniform microparticles with desired properties by carefuillyoptimized procedures prior to adding the fluorescent dye. This alsopermits more flexibility in adjusting the relative concentrations of thedyes. Furthermore, the dyes can be distributed throughout the particlesat higher concentrations than practically attainable by covalentchemical attachment. Such methods are known in the art and generallydescribed in U.S. Pat. No. 6,268,222 which is incorporated herein byreference.

For example, the lipid soluble rhodamine dyes, being freely soluble inorganic solvents, such as ethyl acetate and dichloromethane, andsparingly soluble in water, are easily introduced by solvent-basedaddition of the dyes to previously manufactured polymeric material.Other suitable solvents for this purpose include aromatic hydrocarbons,such as benzene, toluene, xylene; chlorinated hydrocarbons such aschloroform and methylene chloride (preferably mixed with an alcohol likemethanol at about 30% so that the beads do not dissolve); pyridine;ethers like dioxane; tetrahydrofuran; esters such as n-butylphthalate,ethyl phthalate, butylacetate; hydrocarbons such as cyclohexane, methylcyclohexane, ethylcyclohexane; carbon disulfide; and nitropropane.

For example, a quantity of microparticles can be soaked in a 1×10⁻⁴ to5×10⁻³ molar solution of dye in ethyl acetate. The concentrationemployed is not critical as long as it is sufficient to imbibe enough ofthe dye into the microparticles for the microparticles to fluoresce.Similarly, the amount of microparticles employed is not critical as longas the microparticles are fully immersed in the solution so that theycan absorb the dye. The microparticles are kept in contact with thesolution for a time sufficient for the microparticles to swell and,thereby, absorb the dyes. Generally, one hour is sufficient. Once themicroparticles have swollen and a sufficient quantity of the dyes hasbeen absorbed, the microparticles are be made to shrink, therebyentrapping the dyes. Shrinking can be done in a number of ways. Forhydrophobic polymers such as polystyrene, shrinking is easilyaccomplished by adding a large quantity (e.g., 5 fold by volume) ofalcoholic solvent to the solution. An illustrative alcoholic solvent forthis purpose is isopropanol. The microparticles are kept at roomtemperature for a time sufficient to insure shrinking. Generally, 30minutes is sufficient. The microparticles are then separated fromsolution by centrifuge or any other known means of separation andsubsequently washed with more alcoholic solvent.

Alternatively, it is often sufficient simply to admix the polymers anddyes in a mutual solvent. Preferred solvents include chlorinatedaliphatic solvents such as dichloromethane, chloroform, and carbontetrachloride. Alternative solvents include aromatic hydrocarbons suchas benzene, toluene, and xylene. Other solvents include pyridine,dioxane, and dimethylformamide. The solvents may be used in their neatform, mixed together, or mixed with other co-solvents to improve themixtures properties such as lowering or raising the volatility orviscosity. The solvent is then removed by, for example, by pouring themixture onto a surface and letting the solvent evaporate.

The fluorescent properties of the microparticles are measured under setconditions and recorded. For example, the fluorescence intensity of 1 mgof beads in 500 ul of water can be measured and recorded. In thismanner, large batches of uniformly-imbibed microparticles with desiredphysical properties, such as size and charge density, can be prepared.

In instances where it is desirable to imbibe the polymer with a mixtureof dyes, the selected dyes for the mixture can be imbibed sequentially.Alternatively, the selected dyes can be imbibed into the polymersimultaneously. The ratio of dyes employed in the mixture will varydepending on the nature of the dyes and the desired combination ofspectral properties.

5.15 Improved Properties

The fluorescent polymeric materials containing the lipid solublerhodamine dyes, when excited by a light source, emit an unusually strongspectral signal with low background noise. The spectral signal isdependent upon the identities, concentration and ratio of the dyes inthe polymer.

Furthermore, hydrophobic polymeric materials containing the lipidsoluble rhodamine dyes exhibit enhanced dye retention in aqueoussolvent. The dyes are not significantly removed from the polymers by thewater-based solvents commonly used in biologic assays as a suspensionmedium. This is accomplished, in part, by the selection of lipophilicsubstituents, often attached to the amine and imminium moieties, thatimpart a lipophilic nature to the rhodamine dyes.

Finally, fluorescent polymeric materials containing the lipid solublerhodamine dyes are highly photo and chemically stable. In fact, some ofthe lipid soluble rhodamines have photostabilities as high as ten timesthat of fluorescein and 100 times that of cyanine.

In summary, the fluorescent polymeric materials of the instant inventionenjoy all of the benefits associated with the use of rhodamine dyeswhile avoiding the detriments that previously made rhodamine dyesundesirable for such applications.

5.16 Additional Substances

The fluorescent polymeric material may also optionally include one ormore other substances absorbed, adsorbed or covalently attached thereto.As stated above, a non-limiting list of such substances includesbiomolecules, drugs, poisons, vitamins, antigens and pathogens(including viruses, fungi and bacteria) as well as molecules that canact as a probe for a biomolecule, such as biotin. Non-limiting examplesof biomolecules include amino acids, polypeptides, glycosolated andunglycosolated proteins, nucleosides, nucleotides, oligonucleotides,polynucletides, nucleic acids, polynucleic acids such as DNA and RNA,carbohydrates, and steroids. Compounds often classed by theirfunctionality rather than structure are also included, e.g., haptens,toxins, antibodies, enzymes and hormones. In addition, the additionalsubstance can be a probe for a biomolecule, such as biotin. Generally,these substances are either covalently attached to the polymer componentof the fluorescent polymeric material, e.g., through the one or more ofthe “activations” discussed above or incorporated non-covalently byabsorption into, or adsorbtion onto, the polymer component. In oneembodiment, one or more of these substances are covalently attached tothe polymer.

5.17 Illustrative Uses

The fluorescent polymeric materials of the invention can be used forpassive or covalent coupling of biological material, i.e., analyte oranalytical reactant, such as haptens, antigens, antibodies, enzymes ornucleic acids and used for various types of analyte assays such asimmunoassays, nucleic acid (DNA or RNA) assays, affinity purification,cell separation and other medical diagnostic, and industrialapplications.

In one aspect of the invention, a mixture of lipid soluble rhodaminedyes and, optionally, additional dyes, are internally incorporated,simultaneously or sequentially, into a polymeric microparticlepopulation to give the microparticle population a unique spectralsignature or “bar code.” In determining the proportion of each of thedyes to be used to generate the bar code, the intensity values for thespectral profiles of each of the dyes (at standard concentrations) aredetermined in solution at an excitation wavelength desired to be used.The intensity values for each spectral profile are added together to geta composite spectral emission profile, and the relative proportions ofeach of the dyes are adjusted to give a composite spectral emission. Theaddition of spectral profiles and the adjustment of relative proportionsis readily simulated on a computer (starting with the standardexperimental values determined in the previous step), and theseproportions are optionally adjusted in practice.

A number of particle populations are created, each characterized by adifferent spectral bar code. The particles can then be activated orotherwise modified so that they have a specific reactivity with one ormore substances to be measured or assayed (analytes) in a clinical ortest sample. Thus, the spectral bar code in each particle populationcorresponds to a different known reactivity. The particle populationscan then be blended in a specified ratio to form a multicolored particlemixture. Imbibed bead mixtures may contain hundreds to thousands offluorescent dye molecules which greatly increases the sensitivity ofassays employing bead labels in comparison to single dye assays.

To achieve truly multiplexed analysis of a plurality of analytes in asingle sample, some form of identifiable marker, for instance a thirdtype of fluorescent dye or a probe for a biomolecule (e.g., biotin)should be provided that is capable of binding the analyte of interest.This marker has two functions, namely, the capacity to react with theanalyte and the capacity to be detected.

In one embodiment, the microparticles containing bound particles arethen separated and analyzed by conventional methods. Alternatively, allof the microparticles can be analyzed at the same time. Once again, themarker, e.g., a fluorescent dye or biotin, identifies the presence of ananalyte on a given microparticle. The microparticle's fluorescent barcode identifies its specific reactivity. In this manner, the inventionassists in the detection, differentiation, separation, quantificationand analysis of analytes in a single mixture.

The particle populations may be analyzed by any fluorescence detectionsystem, including visual inspection. However, automated techniques arepreferred. Fluorescently imbibed microparticles are particularly wellsuited for flow cytometry analysis. An ordinary flow cytometer iscapable of analyzing spectral properties (fluorescent signals) of up to20,000 particles per second and can provide reliable quantitative dataon a real-time scale.

Spectral differences that may be observed include, but are not limitedto, a difference in excitation maxima, a difference in emission maxima,a difference in fluorescence lifetimes, a difference in fluorescenceemission intensity at the same excitation wavelength or a differentwavelength, a difference in absorption, a difference in fluorescencepolarization, a difference in fluorescence enhancement in combinationwith target materials, or combinations thereof.

The analyte and analyte reactant pairs that may be employed vary widely.For example, the analyte and analyte reactant may be selected from anyof the following combinations wherein either member of the combinationmay be the analyte or the binding partner: antigen and specificantibody; hormone and hormone receptor; hapten and anti-hapten;polynucleotide and complementary polynucleotide; polynucleotide andpolynucleotide binding protein; biotin and avidin or streptavidin;enzyme and enzyme cofactor; and lectin and specific carbohydrate.

The haptens include naturally occurring hormones, naturally occurringdrugs, synthetic drugs, pollutants, allergens, affector molecules,growth factors, chemokines, cytokines, lymphokines, amino acids,oligopeptides, chemical intermediates, nucleotides, oligonucleotides orthe like. The use for such compounds may be in the detection of drugs ofabuse, therapeutic dosage monitoring, health status, donor matching fortransplantation purposes, pregnancy (e.g., hCG or alpha-fetoprotein),detection of disease, e.g., endotoxins, cancer antigens, pathogens, andthe like. Therapeutic drugs may include, but are not limited to,anti-AIDS substances, anti-cancer substances, antibiotics, anti-viralsubstances, enzyme inhibitors, neurotoxins, opioids, hypnotics,antihistamines, tranquilizers, anti-convulsants, muscle relaxants andanti-Parkinson substances, anti-spasmotics and muscle contractants,miotics and anti-cholinergics, immunosuppressants (e.g., cyclosporine)anti-glaucoma solutes, anti-parasite and/or anti-protozoal solutes,anti-hypertensives, analgesics, anti-pyretics and anti-inflammatoryagents (such as NSAID's), local anesthetics, ophthalmics,prostaglandins, anti-depressants, anti-psychotic substances,anti-emetics, imaging agents, specific targeting agents,neurotransmitters, proteins and cell response modifiers. Proteins are ofinterest in a wide variety of diagnostics, such as detecting cellpopulations, blood type, pathogens, immune responses to pathogens,immune complexes, saccharides, lectins, naturally occurring receptors,and the like. Receptors may find use in binding to haptens, proteins,other receptors, or the like, or detection of the presence of pathogens,the level of a particular protein in a physiological fluid, the presenceof haptens in a wide variety of samples, such as physiological fluids,air, process streams, water, etc. Nucleic acids may also find use in thedetection of complementary strands, proteins specifically binding tonucleic acids and the like.

The analyte reactant can be also be selected among fluorescent reportermolecules capable of reacting with a variety of inorganic analytes thatdefine properties of biological fluids, air, and the like, e.g., O₂,CO₂, Ca⁺⁺, Na⁺, K⁺, or Cl⁻.

Of particular interest is the binding of microorganisms and cells,including viruses, prokaryotic and eukaryotic cells, unicellular andpolycellular organism cells, e.g., fungi, animal, mammal, etc., orfragments thereof. Usually, these large aggregations will benon-covalently bound to the surface through specific binding pair membercomplexes. By having a high density of binding members bound to thesurface, a cell or virus may be complexed by a large number of bindingpair members, providing very strong anchoring of the cell, virus, orfragment. The system may then be subjected to vigorous treatment withoutconcern for dislodging the specifically bound entity, whilenon-specifically bound materials may be readily removed.

The fluorescent polymeric materials of the invention may also be usedfor detecting pathogens. Monoclonal antibodies may be linked to thesurface to serve as antibodies. The sample would then be added and cellshaving the epitope recognized by the antibody would bind to the antibodyon the surface. Non-specifically bound pathogens are washed away leavingsubstantially only specifically bound ones. Labeled monoclonalantibodies are then added which are specific for an epitope other thanthe epitope recognized by the catching antibody. The term epitope issynonymous to term antigenic determinant and as used herein means adefined domain on the molecule that serves as a reaction or bindingsite. A molecule may have more than one epitope. For example, firstepitope would allow coupling of the analyte with respective analyticalreactant and second epitope will provide a binding site or domain forthe labeling reagent. In contrast, a competitor molecule will beinterfering (competing) with the formation of a binding pairanalyte-analytical reactant. After incubating to allow reaction betweenthe antibodies and pathogens, non-specifically bound antibodies arewashed away and the presence of the label determined according tostandard detection methods. Non-limiting examples of pathogens ofinterest include Herpesviruses, Poxviruses, Togaviruses,Orthomyxoviruses, Paramyxoviruses, Rhabdoviruses, Coronaviruses,Arenaviruses, and Retroviruses. They may also include bacteria includingbut not limited to Escherichia coli, Pseudomonas aeruginosa,Enterobacter cloacae, Staphylococcus aureus, Enterococcus faecalis,Klebsiella pneumoniae, Salmonella typhimurium, Staphylococcusepidermidis, Serratia marcescens, Mycobacterium bovis, methicillinresistant Staphylococcus aureus and Proteus vulgaris.

Assays using particles of the invention can be carried out in abiological fluid, including separated or unfiltered biological fluidssuch as urine, cerebrospinal fluid, pleural fluid, synovial fluid,peritoneal fluid, amniotic fluid, gastric fluid, blood, serum, plasma,lymph fluid, interstitial fluid, tissue homogenate, cell extracts,saliva, sputum, stool, physiological secretions, tears, mucus, sweat,milk, semen, vaginal secretions, fluid from ulcers and other surfaceeruptions, blisters, and abscesses, and extracts of tissues includingbiopsies of normal, malignant, and suspect tissues or any otherconstituents of the body which may contain the analyte of interest.Other similar specimens such as cell or tissue culture or culture brothare also of interest. Alternatively, the sample is obtained from anenvironmental source such as soil, water, or air; or from an industrialsource such as taken from a waste stream, a water source, a supply line,or a production lot. Industrial sources also include fermentation media,such as from a biological reactor or food fermentation process such asbrewing; or foodstuff, such as meat, game, produce, or dairy products.The test sample can be pre-treated prior to use, such as preparingplasma from blood, diluting viscous fluids, or the like; methods oftreatment can involve filtration, distillation, concentration,inactivation of interfering compounds, and the addition of reagents.

5.18 Inherent Limitations In Structures

Those skilled in the art will appreciate that many of the lipid solublerhodamine dye compounds described in the various structures herein mayexhibit the phenomena of tautomerism, conformational isomerism,geometric isomerism and/or stereo isomerism. As the structures presentedin the specification and claims can represent only one tautomeric,conformational isomeric, enantiomeric or geometric isomeric form, itshould be understood that the invention encompasses any tautomeric,conformational isomeric, enantiomeric and/or geometric isomeric forms ofthe compounds that have one or more of the utilities described herein.As a specific example, reference is made throughout the specification tothe C3 amino and C6 imminium substituents. As this nomenclaturecorresponds to the illustrated structures, which represent only one ofseveral possible tautomeric forms (or resonance structures) of thecompounds, it will be understood that these references are forconvenience only and that any such references are not intended to limitthe scope of the compounds described herein.

Furthermore, those of skill in the art will recognize that the lipidsoluble rhodamine dyes of the invention may exist in many differentprotonation states, depending on, among other things, the pH of theirenvironment. The structures provided herein depict the compounds in onlyone of several possible protonation states. Accordingly, it will beunderstood that these structures are illustrative only, and that theinvention is not limited to any particular protonation state—any and allprotonated forms of the dyes are intended to fall within the scope ofthe invention.

As the lipid soluble rhodamine dye compounds used in the invention maybear positive charges, depending upon their physical state, they oftenhave counterions associated therewith. The identity or identities of anyassociated counterions is typically dictated by the synthesis and/orisolation methods by which the compounds are obtained. Typicalcounterions include, but are not limited to, halides, acetate,trifluoroacetate, any salt of a strong acid, and mixtures thereof. Itwill be understood that the identity or identities of any associatedcounterion's is not a critical feature of the invention and that theinvention encompasses the use of dyes in association with any type ofcounter ion. Moreover, as the compounds can exists in a variety ofdifferent forms, the invention is intended to encompass not only formsof the dyes that are in association with counterions (e.g., dry salts),but also forms that are not in association with counterions (e.g.,aqueous or organic solutions).

5.19 Incorporation By Reference

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by reference.No admission is made that any reference cited in this specification isprior art.

6. EXAMPLES 6.1 Experimental Overview

Lipid soluble rhodamine dyes were synthesized from reactions ofsubstituted and un-substituted 1-hydroxy-3 amino-benzene derivativeswith phenyl aldehyde derivatives (Examples 1, 2, and 3; FIGS. 1, 2, and3). In general, the N-alkyl-1-hydroxy-3-amino-benzene intermediates weregenerated from N-alkyl-1-amino-3-methyoxybenzene derivatives. TheN-alkyl 1-amino-3-methyoxybenzene derivatives were synthesized byN-alkylation of 1-amino-3-methyoxybenzene derivatives. In general,phenyl aldehyde derivatives and amino-3-methyoxybenzene derivatives wereavailable from commercial sources (Aldrich Chemical company).Alternatively, lipid soluble rhodamine dyes were synthesized by asecondary derivatization of dyes first synthesized by established dyeforming procedures (Example 4 and Example 5).

Following this general procedure, in accordance with the more detailedexample descriptions below, the following dyes were obtained:

Dye structures were verified by mass spectrometry (see Table 1 below).

6.2 Dye Synthesis

The general synthetic procedure for synthesis of bis N,N-dialkyllipophilic rhodamine dyes of structure 140 is outlined in FIG. 1.4-methyl-3-amino-1-methoxybenzene 110 (R′=R′″=H, R″=Me) was suspended ina neat solution of n-dodecylbromide in the presence of excess (10equivalents) anhydrous KHCO₃ powder. The reaction was heated at 130° C.for 24 hours. The cooled reaction mixture was filtered through a plug ofsilica gel eluting with hexane and then ethyl acetate/hexane (1:20) toyield pure 4 methyl-3-dodecylamino-1-methoxy benzene 120 (R=Me, R′=R′″H, R″=Me) in 70% yield (¹HNMR (CDCl₃) δ 7.02 (d, 1H), 6.27 (d, 1H), 3.86(s, 3H), 3.54 (bs, NH), 3.20 (t, 2H), 2.14 (s, 3H), 1.74 (p, 2H), 1.41(m, 18H), 0.98 (t, 3H))

As a general methyl group de-protection procedure, 4methyl-3-dodecylamino-1-methoxy benzene 120 was suspended in dry CH₂Cl₂,cooled to 0° C. and 5 equivalents of a solution of BBr₃ (1 M CH₂Cl₂,Aldrich Chem. Co.) were added and the reaction maintained at 0° C. for16 hours. The reaction was quenched at 0° C. by slow addition of MeOHand then careful addition of solid NaHCO₃ (10 Equiv.) until foamingstops. The mixture was filtered, the filtrate was concentrated to anoil, and the product was isolated by normal phase chromatography elutingwith methanol/CH₂Cl₂. The solvent was removed from the pure fractions togive 3-dodecylamino-1-hydroxy-4 methylbenzene 120 (R=H, R′=R′″=H, R″=Me)as a waxy solid in 70-80% yield (¹HNMR (CDCl₃) δ 6.91 (d, 1H), 6.17 (d,1H), 6.14 (dd, 1H) 4.62 (bs, OH), 3.53 (bs, NH), 3.14 (t, 2H), 2.09 (s,3H), 1.70 (p, 2H), 1.46 (m, 18H), 0.93 (t, 3H))

As a general dye synthesis procedure, 3-dodecylamino-1-hydroxy-4methylbenzene 120 (R=H) and a phenyl aldehyde of general structure 130were suspended in dichlorobenezene and the reaction mixture was heatedwith stirring at 155° C. for 1 hour. Different phenyl aldehydes werefound to require different temperatures and reaction times to produceoptimal yields in the dye forming reaction. For example; whenR¹¹=R¹⁵=chlorine, a 30 minute reaction time at 145° C. resulted incomplete conversion of starting materials, and when R¹¹-R¹⁵=Fluorine areaction time of 20 minutes at 135° C. was sufficient for completeconversion. The reaction mixture was allowed to cool and solid LiClO₄was added with stirring. The resulting mixture was suspended in CH₂Cl₂and loaded directly unto a small pack of silica gel. The silica pack wasfirst eluted with CH₂Cl₂ and then with methanol/dichloromethane (1:99).The fractions containing dye were collected and concentrated to an oil.

Thus dyes 1, 3, and 5 of general structure I (where R⁴=R⁵=R¹=R⁸=H, andR²=R⁷=Me) were produced by the general procedure in reactions of 120(R=H, R′=R′″=H, R″=Me) with compounds 130 where R″ to R″=H, for Dye 1,R¹¹=R¹⁵=chlorine and R¹² to R¹⁴=H for Dye 3, R¹¹=R¹⁵=fluorine and R¹² toR¹⁴=H for Dye 5.

6.3 Dye Synthesis

The general synthetic procedure for synthesis of N,N, N′,N′-tetraalkyllipophilic rhodamine dyes of structure 240 is outlined in FIG. 2.3-amino-1-methoxybenzene 110 was suspended in a neat solution ofn-dodecylbromide in the presence of excess (10 equivalents) anhydrousKHCO₃ powder. The reaction was heated at 130° C. for 72 hours. Thecooled reaction mixture was filtered through a plug of silica geleluting with hexane and then ethyl acetate/hexane (1:20) to yield pure3-(N,N-didodecylamino-1-methoxy benzene 220 (R=Me, R′=R″=R′″=H) in 70%yield (¹HNMR (CDCl₃) δ 7.15 (t, 1H), 6.31 (d, 1H), 6.24 (m, 2H), 3.83(s, 3H), 3.27 (t, 4H), 1.60 (p, 4H), 1.34 (m, 36H), 0.93 (t, 6H))

By the general de-protection procedure described for 120, methyl groupde-protection of 220 (R=Me) gave 3-(N,N-didodecylamino-1-hydroxy-4methylbenzene 220 (R=H, R′=R″=R′″=H) as a waxy solid in 70-80% yield(¹HNMR (CDCl₃) δ7.08 (t, 1H), 6.27 (dd, 1H), 6.16 (t, 1H), 6.13 (dd,1H), 4.61 (bs, OH), 3.25 (t, 4H), 1.61 (p, 4H), 1.33 (m, 36H), 0.93 (t,6H)).

By the general dye synthesis procedure described for 140,3-(N,N-didodecylamino-1-hydroxy-4 methylbenzene 220 reacted with phenylaldehydes 130 to give dyes of general structure 240. As in synthesis ofdye 140, different phenyl aldehydes required different temperatures andreaction times to produce optimal yields in the dye forming reactions.

Thus, dyes 2 and 7 of general structure I (where R¹=R²=R⁴=R⁵=R⁷=R⁸=H)were produced by the reactions from starting materials 220 (R=H,R′=R″=R′″=H) and 130 where R¹¹ to R¹⁵=H for Dye 2, R¹¹=R¹⁵=chlorine andR¹¹ to R¹⁵=fluorine for Dye 7.

6.4 Dye Synthesis

The general synthetic procedure for synthesis of N-alkyl indolinelipophilic rhodamine dyes of structure 340 is outlined in FIG. 3.3-Amino-1-methoxybenzene 110 (R″=Me, R′″=H) was mixed with isobutyrylbromide in CH₂Cl₂ and 3 equivalents of triethylamine were addeddrop-wise at 0° C. The reaction mixture was stirred for 2 hours,quenched by addition of dilute H₂SO₄, and the organic layer wasseparated and dried with MgSO₄. Purification by normal phasechromatography eluting with hexane:CH₂Cl₂ (1:1) gave 111 in 96% yield.The intermediate 111 was mixed with 5 equivalents AlCl₃ and heated at140° C. for 2 hours. The mixture was cooled and the solid trituratedwith CH₂Cl₂. The solution was concentrated to an oil and then suspendedin acetone. To the solution was added 10 equivalents methyl iodide and15 equivalents K₂CO₃. The reaction was stirred overnight, filtered, andconcentrated to an oil. Purification by normal phase chromatographyeluting with hexane: CH₂Cl₂ (0.3:1) gave 112 in 60% yield. Intermediate112 was suspended in tetrahydrofuran and the solution was added to asuspension of 5 equivalents of lithium aluminum hydride intetrahydrofuran. The mixture was refluxed for 1.5 hours and then allowedto cool. The reaction was quenched at by slow addition of ice chips andthen cold 1 M NaOH. The mixture was transferred to a separatory funneland partitioned into ethyl acetate. The organic layer was dried withNa₂SO₄ and concentrated to an oil. The crude mixture was separated bynormal phase chromatography eluting with hexane/ethyl acetate (1:10) togive 310 in 21% yield

Following the general procedures outlined for synthesis of 120 from 110,N-alkylation of 310 with n-dodecylbromide and methyl group deprotectionproduced 320 (320 R=H, R″=Me, R′″=H; H¹NMR CDCl₃ 6.72 (d, 1H), 6.08 (d,1H), 4.65 bs, OH), 3.27 (t, 2H), 3.22 (s, 2H), 2.29 (s, 3H), 1.59 (p,2H), 1.45 (s, 6H), 1.35 (m, 18H), 0.94 (t, 3H))

Following the general dye synthesis procedure described for 140,Lipophilic rhodamine dyes of structure 340 were synthesized. Thus,N-dodecyl-hydroxy-methylindoline 320 was reacted with phenyl aldehydes130 and the fractions containing 340 were collected and concentrated tooils. By analogy to synthesis of dye 140, different phenyl aldehydesrequired different temperatures and reaction times to produce optimalyields in the dye forming reactions. Thus, Dyes 9 and Dye 10 of generalstructure I (where R⁷=R¹=Me, R¹=R⁸=H) were produced from startingmaterials 320 ((R=H, R″=Me, R′″=H) and 130 where R¹¹ to R¹⁵=H for Dye 9,and R¹¹ to R¹⁵=fluorine for Dye 10.

6.5 Dye Synthesis

Lipophilic rhodamine dyes of the general structure 140 and 240, whereone or more of R¹¹ to R¹⁵ equal carboxylate ester, were synthesized fromthe carboxylate dye intermediates which had been first synthesized byestablished procedures. In general, carboxylate substituted dyes wereproduced by reaction of trimelletic acid anhydride derivatives orphthallic anhydride derivatives with aminohydroxybenzene derivatives,such as 120, 220, or 320 in methanesulfonic acid with heating.

The carboxylate acid groups were esterified by reaction of the dye acidwith the alcohol under standard acid catalyzed esterificationconditions. Thus, Dye 6 was generated by reaction of 220 (R=H, R″=Me,R′=R′″=H) with phthallic anhydride followed by esterification withoctadecyl alcohol (Dye 6; 240 where R¹¹=octadecyl carboxy ester, R¹² toR¹⁵=H, R¹=R²=R⁷=R⁸=H).

6.6 Dye Synthesis

Lipophilic rhodamine dyes of general structure (II) (where R⁹=H) weresynthesized by secondary derivatization of carboxylate substituted dyesfirst synthesized according established procedures as illustrated, forexample, in U.S. Pat. No. 5,936,087 (Benson et al.). In general, dyes ofgeneral structure (II) were produced by reaction of DMF and POCL₃ withaminohydroxybenzene derivatives of general structure 310 (where R=Me, R″to R″ bridged benzo). N-Aryl or N-alkyl carboxylate derivatives of 310were synthesized by established procedures. Amidation of carboxysubstituted dyes of general structure (II) was typically performed byNHS ester activation and reaction with the preferred amine. Thus, Dye 15was generated by reaction of the Dye NHS with dodecylamine.

6.7 Spectral Analysis

Concentrated stocks of the dye were made by suspending the dye in ethylacetate or CH₂Cl₂. Absorption spectra of equal aliquots of the dyes wererecorded (HP model 8451A diode array spectrophotometer) in methanol andtoluene, and the spectra were monitored for changes in absorptionintensity and wavelength. In general, dyes with good spectral propertiesin non-polar organic solvents, such as toluene, were considered goodcandidates for polystyrene imbibing. An extinction coefficient equal to80,000 for the dyes in methanol was used to approximately determine thedye concentrations for fluorescence measurements and bead imbibingexperiments. Fluorescence emission and excitation spectra were recordedfor diluted dye stocks in methanol and toluene at equal concentrationsof the dyes (0.6×10⁻⁶ M).

TABLE 1 Spectral Analysis Emission Emission λmax Emission Mass Spectrumλmax Toluene λmax Beads Dye Calc./Observed MeOH (nm) (nm) (nm) 1650.52/[M + H]⁺ 651.6 553 560 548 2  959.9/[M]⁺ 960.0 586 586 586 3718.44/[M + H]⁺ 719.52 564 575 579 5  686.5/[M + H]⁺ 687.6 562 579 573 61256.1/[M]⁺ 1256.21 588 596 587 7 1049.8/[M]⁺ 1050.0 613 612 612 9759.62/[M]⁺ 759.63 615 626 616 10 849.57/[M]⁺ decomp 661 657 659 15841.46/[M + H]⁺ 842.60 652 655 654

6.8 Bead Coding Assays

A general procedure was followed to imbibe polystyrene beads with thelipophilic rhodamine dyes 1, 2 and 9, both individually and incombination. The polystyrene beads were typically 1-10 um in diameter,preferably 5 um in diameter, and contained from 1 to 20%, preferablyfrom 1 to 5%, crosslinking. Such beads are commercially available from anumber of sources (e.g., Bangs labs, Dynal, Spereotech, Polyscience).The dye stocks were set at 1×10⁻³ molar in ethyl acetate. Threemilligrams of dry beads were weighed into a eppendorf tube. Differentamounts of dye stocks were diluted into ethyl acetate, either as amixture or as a single dye. The diluted ethyl acetate solutions wereadded at once to the beads. The beads were kept in contact with the dyesolutions for 1 hour and then 5 fold volume of isopropanol was added tothe bead suspension. The beads were kept at room temperature for anadditional 30 minutes and then the tube was centrifuged down and the dyesolution separated from the beads. The beads were washed withisopropanol. The fluorescence intensity of the beads was recorded for a1/3 dilution of the beads in water suspended in 500 ul of water (1mg/500 ul). FIG. 4 shows the emission spectrum of beads imbibed with amixture of the three dyes in comparison to beads imbibed with each dyealone and the predicted sum of the three single dye spectra.

6.9 Fluorescent Reference Standards

Three grams of polystyrene plastic are dissolved in 10 ml of a suitableneutral organic solvent with agitation. Preferred solvents includechlorinated aliphatic solvents such as dichloromethane, chloroform, andcarbon tetrachloride. Alternative solvents include aromatic hydrocarbonssuch as benzene, toluene, and xylene. Other solvents include pyridine,dioxane, and dimethylformamide. The solvents may be used in their neatform, mixed together, or mixed with other co-solvents to improve themixtures properties such as lowering or raising the volatility orviscosity. A lipophilic rhodamine dye is suspended in thedichloromethane or another preferred solvent to give a concentratedstock solution. Usually the dye is suspended in the same solvent as thatused to dissolve the polystyrene. The stock concentration is determinedspectrophotometrically. An aliquot of the concentrated stock is mixedinto a portion of the pre-mixed polystyrene solution to give the desireddye concentrations of from 1×10-3 to 1×10-15 M, preferably 1×10-6 M to1×10-9 M. The dye polystyrene mixture is pipetted or poured into a moldor spotted onto a surface and let sit from 5 to 24 hours until thesolvent evaporates. If desired, the resultant solid dyed polystyrene canalso be machined. The solid dyed polystyrene is then used as afluorescent reference standard for testing fluorescence intensity orspectral shape in quality control analysis of fluorescence detectionbased instruments. Similarly, the dyed polystyrene can be formed inwells of microtiter plates and fluorescence measurements used toestablish well location or set focal depths of fluorescence basedinstruments.

1. A fluorescent polymeric material comprising a polymer and at leastone lipid-soluble rhodamine dye comprising one of the following corestructures:

where R¹ R², R^(2′), R^(3′), R^(3″), R⁴, R^(4′), R^(5′), R⁵, R^(6″),R^(6′), R^(7′), R⁷, R⁸, R⁹, R¹¹, R¹² R¹², R¹³, R¹⁴ and R¹⁵ are,independently, selected from hydrogen or the same or differentsubstituent groups having no more than 40 atoms, where at least one ofR^(3′), R^(3″), R^(6″) and R^(6′) is a lipophilic substituent; where R⁹in core structure (II) is a nonaromatic substituent; where, with respectto core structures (I), (III) and (IV), if R^(3″) and R^(6″) are eachphenyl or substituted phenyl and R^(3′) and R^(6′) are each hydrogenthen at least two of R¹¹, R¹², R¹³, R¹⁴ and R¹⁵ must be other thanhydrogen; and where, with respect to core structures (I), (III) and(IV), R¹ and R⁸ are not, simultaneously, pendant or fused benzo, napthoor polycyclic aryleno groups.